High thebaine poppy and methods of producing the same

ABSTRACT

This disclosure relates to the production of opium poppy plants having high levels of thebaine. More particularly, the disclosure relates to the production of opium poppies having high levels of thebaine by simultaneously reducing the expression of genes encoding thebaine 6-0-demethylase (T60DM) and codeine 3-0-demethylase (CODM).

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/762,090, filed Mar. 21, 2018, which is a U.S. National PhaseApplication of PCT International Application No. PCT/CA2017/050951,filed Aug. 11, 2017, which is an International Application of and claimsthe benefit of priority to Canadian Patent Application No. 2,941,315,filed Sep. 7, 2016, and U.S. Patent Application No. 62/374,682, filed onAug. 12, 2016, the entire contents of which are herein incorporated byreference.

BACKGROUND OF THE DISCLOSURE 1. Field of Disclosure

This disclosure relates to the production of opium poppies having highlevels of thebaine. More particularly, the disclosure relates to theproduction of opium poppies having high levels of thebaine bysimultaneously reducing the expression/activity of thebaine6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM).

2. Reference to a “Sequence Listing,” a Table, or a Computer ProgramListing Appendix Submitted on a Compact Disk

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is Sequence_Listing_050478_501C01US. The text fileis 32.6 KB, was created on Apr. 21, 2021, and is being submittedelectronically via EFSWeb.

3. Description of Related Art

Opioids are psychoactive substances derived from the opium poppy(Papaver somniferum), or their synthetic analogues. Opioids have thepotential to cause substance dependence that is characterized by astrong desire to take opioids, impaired control over opioid use,persistent opioid use despite harmful consequences, a higher prioritygiven to opioid use than to other activities and obligations, increasedtolerance, and a physical withdrawal reaction when opioids arediscontinued. As of 2014, there were an estimated 17 million people whosuffer from opioid dependence (i.e. an addiction to opioids). Themajority of people dependent on opioids use illicitly cultivated andmanufactured heroin. Due to their pharmacological effects, opioids inhigh doses can cause respiratory depression and death. As of 2014, anestimated 69 000 people die worldwide from opioid overdose each year.

In the World Drug Report 2016, the United Nations Office on Drugs andCrime indicated that recent declines in opium production would not leadto major shortages in the global heroin market given the high opiumproduction levels of previous years. Thus, it may take a period ofsustained decline in opium production for the repercussions to be feltin the heroin market. It would be desirable to have a method ofdisrupting opium production by turning off poppy plant genes necessaryfor the production of psychoactive alkaloids in the field.

Hydroxymorphinans, such as oxycodone, naloxone, naltrexone, nalbuphineand nalmefene are important opiate derivatives due to their utility aspotent analgesics and/or narcotic antagonists. The most practicalsynthetic routes to the preparation of these pharmaceuticals use thealkaloid, thebaine, as a starting material. Other important opiatederivatives such as the ring-C bridged compounds buprenorpine andetorphine are also most practically prepared from thebaine.

Unfortunately, thebaine is costly due to its limited availability. Totalsynthesis is difficult and, in poppy plants, thebaine typicallyaccumulates to low levels of only 0.5 to 2% of the total alkaloids inopium poppy. Referring to FIG. 1, thebaine exists at a branch point ofmorphine biosynthesis, being the substrate for two competing enzymes.Thebaine 6-O-demethylase (T6ODM) converts thebaine to oripavine, andcodeine 3-O-demethylase (CODM) converts thebaine to neopinone.

Mutants of opium poppy accumulating thebaine and oripavine rather thanmorphine and codeine have been reported, including theTOP1 varietyderived through chemical mutagenesis (Millgate et al. 2004). Althoughthe metabolic block in TOP1 was suggested to result from a defect in theenzyme catalyzing the 6-O-demethylation of thebaine and oripavine, thebiochemical basis for the phenotype was not determined. Moreover, amicroarray was used to identify 10 genes underexpressed in TOP1, whichlist did not include any enzymes theoretically capable ofO-demethylation. A plant line containing the TOP1 mutation was depositedunder the Budapest Treaty with the American Type Culture Collection onMar. 20, 2008, under ATCC Patent Deposit Designation PTA-9110.WO2009/109012 discloses the mutagenesis of the line designated PTA-9110to produce a further line accumulating high levels of thebaine, whichwas deposited under the Budapest Treaty with the American Type CultureCollection on Mar. 20, 2008, under ATCC® Patent Deposit DesignationPTA-9109. However, the biochemical basis for the phenotype was notexplored.

Researchers have been interested in using molecular approaches toengineer opium poppy to produce opioids of choice for several years,however, the results have at times been unexpected and frustrating. Forexample, Allen et al. (Nature Biotechnology 22:1559-1556) used RNAinterference (RNAi) to silence the genes encoding codeinone reductase(COR), the penultimate enzyme of morphine biosynthesis. COR convertscodeinone to codeine. However, rather than resulting in the accumulationof codeinone, elimination of COR activity resulted in accumulation ofreticuline, i.e. seven enzymatic steps before COR. The surprisingaccumulation of reticuline suggests a feedback mechanism preventingintermediates from general benzylisoquinoline synthesis entering themorphine-specific branch.

SUMMARY

This disclosure relates to the production of opium poppies having highlevels of thebaine. More particularly, the disclosure relates to theproduction of opium poppies having high levels of thebaine bysimultaneously reducing the expression/activity of thebaine6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM).

Various aspects of the disclosure relate to a method of increasingaccumulation of thebaine in an opium poppy plant, the method comprisinggenetically modifying the plant to simultaneously reduce the activity ofthebaine 6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM) inthe poppy plant. The wild type T6ODM may have the amino acid sequence ofSEQ ID NO: 1. The wild type CODM may have the amino acid sequence of SEQID NO: 3.

In some instances, genetically modifying the plant to simultaneouslyreduce the activity of T6ODM comprises introducing an expressionconstruct to reduce the accumulation of transcripts from an endogenousgene encoding T6ODM. In some instances, the sequence of the expressionconstruct to reduce the accumulation of transcripts from the endogenousgene encoding T6ODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4In some instances, genetically modifying the plant to simultaneouslyreduce the activity of T6ODM comprises introducing a loss of functionmutation in an endogenous gene encoding T6ODM.

In some instances, genetically modifying the plant to simultaneouslyreduce the activity of CODM comprises introducing an expressionconstruct to reduce the accumulation of transcripts from an endogenousgene encoding CODM. In some instances, the sequence of the expressionconstruct to reduce the accumulation of transcripts from the endogenousgene encoding CODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4.In some instances, genetically modifying the plant to simultaneouslyreduce the activity of CODM comprises introducing a loss of functionmutation in an endogenous gene encoding CODM.

Various aspects of the disclosure relate to a method of producing anopium poppy plant with increased levels of thebaine relative to a wildtype plant, the method comprising: crossing a first parent having atleast one loss of function allele of the gene encoding thebaine6-O-demethylase with a second parent having at least one loss offunction allele of the gene encoding codeine 3-O-demethylase; andallowing progeny that have both the loss of function mutation allele ofthe gene encoding thebaine 6-O-demethylase and the loss of functionallele of the gene encoding codeine 3-O-demethylase to self pollinate toproduce a plant that is homozygous for the loss of function mutationallele of the gene encoding thebaine 6-O-demethylase and homozygous forthe loss of function allele of the gene encoding codeine3-O-demethylase.

Various aspects of the disclosure relate to a method for producing anopium poppy plant with increased thebaine content, the methodcomprising: decreasing the expression an endogenous gene encoding anendogenous thebaine 6-O-demethylase (T6ODM) in the plant; and decreasingthe expression of an endogenous gene encoding codeine 3-O-demethylase(CODM) in the plant. In some instances, decreasing the expression of theendogenous gene encoding T6ODM comprises introducing or producing a lossof function allele in the endogenous gene encoding T6ODM. In someinstances, decreasing the expression of the endogenous gene encodingCODM comprises introducing or producing a loss of function allele in theendogenous gene encoding CODM.

In some instances, decreasing the expression of the endogenous geneencoding T6ODM comprises expressing a first heterologous nucleic acidmolecule homologous to a portion of the endogenous gene encoding T6ODM,wherein the first heterologous nucleic acid molecule decreasesexpression of the endogenous gene encoding T6ODM. In some instances,decreasing the expression of the endogenous gene encoding CODM comprisesintroducing or producing a loss of function allele in the endogenousgene encoding CODM. In some instances, decreasing expression of theendogenous gene encoding CODM comprises expressing a second heterologousnucleic acid molecule homologous to a portion of the endogenous geneencoding CODM, wherein the second heterologous nucleic acid moleculedecreases expression of the endogenous gene encoding CODM.

In some instances, decreasing expression of the endogenous gene encodingCODM comprises expressing a heterologous nucleic acid moleculehomologous to a portion of the endogenous gene encoding CODM, whereinthe heterologous nucleic acid molecule decreases expression of theendogenous gene encoding CODM. In some instances, decreasing T6ODMactivity comprises introducing or producing a loss of function allele inthe endogenous gene encoding T6ODM.

In some instances, the loss of function allele comprises a disruption orpoint mutation in the gene. The disruption may be a deletion or aninsertion. An insertion may be a T-DNA or a transposable element.

In some instances, the first heterologous nucleic acid moleculedecreases expression of the endogenous gene encoding T6ODM by RNAinterference.

In some instances, the second heterologous nucleic acid moleculedecreases expression of the endogenous gene encoding CODM by RNAinterference.

In some instances, the heterologous nucleic acid molecule decreasesexpression of the endogenous gene encoding CODM by RNA interference.

In some instances, the heterologous nucleic acid molecule comprises aportion of SEQ ID NO: 2 or SEQ ID NO: 4. In some instances, theheterologous nucleic acid molecule comprises a portion of SEQ ID NO: 7.In some instances, the heterologous nucleic acid molecule comprises aportion of SEQ ID NO: 8.

In some instances, the T6ODM has an amino acid sequence at least 95%identical to SEQ ID NO: 1 and the CODM has an amino acid sequence atleast 95% identical to SEQ ID NO: 3.

Various aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plan, the method comprising: i) using a molecularmethodology to identify a first plant as comprising a loss of functionallele in an endogenous gene encoding codeine 3-O-demethylase (CODM);ii) establishing a cross of said first plant to a second plant having aloss of function allele in an endogenous gene encoding thebaine6-O-demethylase (T6ODM); iii) allowing progeny from the cross toself-fertilize; and iv) screening progeny from self-fertilized plantsfor a plant that is homozygous for both the loss of function allele inthe endogenous gene encoding CODM and the loss of function allele in theendogenous gene encoding T6ODM.

In some instances, the second plant having the loss of function allelein the endogenous gene encoding T6ODM is identified using a molecularmethodology. In some instances, the loss of function allele in theendogenous gene encoding T6ODM is generated by genetic modification ofthe second plant or an ancestor thereof. In some instances, the secondplant having the loss of function allele in the endogenous gene encodingT6ODM is a plant of the line deposited as ATCC PTA-9110.

Various aspects of the disclosure relate to a method of generating anopium poppy plant having an thebaine content relative to a wild typeopium poppy plant, the method comprising: i) using a molecularmethodology to identify a first plant as comprising a loss of functionallele in an endogenous gene encoding thebaine 6-O-demethylase (T6ODM);ii) establishing a cross of said first plant to a second plant having aloss of function allele in an endogenous gene encoding codeine3-O-demethylase (CODM); iii) allowing progeny from the cross toself-fertilize; and iv) screening progeny from self-fertilized plantsfor a plant that is homozygous for both the loss of function allele inthe endogenous gene encoding CODM and the loss of function allele in theendogenous gene encoding T6ODM. In some instances, the second planthaving the loss of function allele in the endogenous gene encoding CODMis identified using a molecular methodology. In some instances, the lossof function allele in the endogenous gene encoding CODM is generated bygenetic modification of the second plant or an ancestor thereof. In someinstances, the second plant having the loss of function allele in theendogenous gene encoding CODM is a plant of the line deposited as ATCCPTA-9109.

Various aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a plant as comprising a loss of function allelein an endogenous gene encoding thebaine 6-O-demethylase (T6ODM); ii)genetically modifying the plant to introduce a loss of function allelein an endogenous gene encoding codeine 3-O-demethylase (CODM); iii)allowing the plant to self-fertilize; and iv) screening progeny from theself-fertilized plant for a plant that is homozygous for both the lossof function allele in the endogenous gene encoding CODM and the loss offunction allele in the endogenous gene encoding T6ODM.

Various aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a plant as comprising a loss of function allelein an endogenous gene encoding codeine 3-O-demethylase (CODM); ii)genetically modifying the plant to introduce a loss of function allelein an endogenous gene encoding thebaine 6-O-demethylase (T6ODM) in theplant by genetic modification;

-   -   iii) allowing the plant to self-fertilize; and iv) screening        progeny from the self-fertilized plant for a plant that is        homozygous for both the loss of function allele in the        endogenous gene encoding CODM and the loss of function allele in        the endogenous gene encoding T6ODM.

Various aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a plant as comprising a loss of function allelein an endogenous gene encoding thebaine 6-O-demethylase (T6ODM); ii)genetically modifying the plant to reduce expression of an endogenousgene encoding codeine 3-O-demethylase (CODM); iii) allowing the plant toself-fertilize; and iv) screening progeny from the self-fertilized plantfor a plant that is homozygous for the loss of function allele in theendogenous gene encoding T6ODM and has reduced expression of theendogenous gene encoding CODM. In some instances, genetically modifyingthe plant to reduce expression of the endogenous gene encoding CODMcomprises introducing an expression construct to express a hairpin RNAtargeting the endogenous gene encoding CODM.

Various aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a plant as comprising a loss of function allelein an endogenous gene encoding codeine 3-O-demethylase (CODM); ii)genetically modifying the plant to reduce expression of an endogenousgene encoding thebaine 6-O-demethylase (T6ODM); iii) allowing the plantto self-fertilize; and iv) screening progeny from the self-fertilizedplant for a plant that is homozygous for both the loss of functionallele in the endogenous gene encoding CODM and has reduced expressionof the endogenous gene encoding T6ODM. In some instances, geneticallymodifying the plant to reduce expression of the endogenous gene encodingT6ODM comprises introducing an expression construct to express a hairpinRNA targeting the endogenous gene encoding T6ODM.

In some instances, the molecular methodology comprises targeting inducedlocal lesions in genomes (TILLING) methodology.

Various aspects of the disclosure relate to an opium poppy plantproduced by a method as described above.

Various aspects of the disclosure relate to a genetically modified opiumpoppy plant or plant cell having reduced activity of thebaine6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM) relative to awild type plant, wherein the opium poppy plant is genetically modifiedto have reduced expression of T6ODM, and CODM, or both.

In some instances, the plant comprises a first expression construct forreducing the expression of T6ODM and a second expression construct forreducing expression of CODM. In some instances, the first expressionconstruct comprises a first nucleic acid molecule encoding a firsthairpin RNA for reducing expression of an endogenous gene encodingT6ODM. In some instances, the endogenous gene encoding T6ODM encodes anmRNA comprising having the sequence of SEQ ID NO: 15. In some instances,the nucleic acid molecule encoding the first hairpin RNA comprises aportion of SEQ ID NO: 2.

In some instances, the second expression construct comprises a secondnucleic acid molecule encoding a second hairpin RNA for reducingexpression of an endogenous gene encoding CODM. In some instances, theendogenous gene encoding CODM encodes an mRNA comprising having thesequence of SEQ ID NO: 16. In some instances, the nucleic acid moleculeencoding the second hairpin RNA comprises a portion of SEQ ID NO: 4.

In some instances, the plant or plant cell comprises an expressionconstruct comprising a nucleic acid molecule for reducing the expressionof T6ODM and CODM. In some instances, the nucleic acid molecule encodesa hairpin RNA for reducing expression of an endogenous gene encodingCODM. In some instances, the nucleic acid molecule encodes a hairpin RNAfor reducing expression of an endogenous gene encoding T6ODM. In someinstances, the nucleic acid molecule encodes a single hairpin RNAsufficient to reduce expression of endogenous genes encoding T6ODM andCODM. In some instances, the nucleic acid molecule comprises a portionof SEQ ID NO:2, SEQ ID NO:4, or both.

In some instances, the expression construct comprises a first nucleicacid molecule encoding a first hairpin RNA for reducing expression of anendogenous gene encoding T6ODM and a second nucleic acid moleculeencoding a second hairpin RNA for reducing expression of an endogenousgene encoding CODM. In some instances, the endogenous gene encodingT6ODM encodes an mRNA comprising having the sequence of SEQ ID NO: 15.In some instances, the endogenous gene encoding T6ODM encodes apolypeptide having the sequence of SEQ ID NO 1. In some instances, theendogenous gene encoding CODM encodes an mRNA comprising having thesequence of SEQ ID NO: 16. In some instances, the endogenous geneencoding T6ODM encodes a polypeptide having the sequence of SEQ ID NO 3.In some instances, each of the first nucleic acid molecule and thesecond nucleic acid molecule comprise a portion of SEQ ID NO: 2, SEQ IDNO: 4, or both.

In some instances, the nucleic acid molecule encoding the hairpin RNA(s)comprises a portion of SEQ ID NO: 8. In some instances, the nucleic acidmolecule encoding the hairpin RNA(s) comprises a portion of SEQ ID NO:7.

In some instances, the first nucleic acid molecule comprises a portionof SEQ ID NO: 8. In some instances, the first nucleic acid moleculecomprises a portion of SEQ ID NO: 7.

In some instances, the second nucleic acid molecule comprises a portionof SEQ ID NO: 8. In some instances, the second nucleic acid moleculecomprises a portion of SEQ ID NO: 7.

In some instances, the plant or plant cell is genetically modified tohave reduced activity of T6ODM, and the reduced activity of CODM isconferred by a mutation in the endogenous gene encoding CODM that wasnot introduced by genetic modification of the plant or plant cell. Insome instances, mutation in the endogenous gene encoding CODM that wasnot introduced by genetic modification of the plant or plant cell is themutation present in seeds of the plant deposited under Patent DepositDesignation PTA-9109.

In some instances, the plant is genetically modified to have reducedactivity of CODM, and wherein reduced activity of T6ODM is conferred bya mutation in the endogenous gene encoding T6ODM that was not introducedby genetic modification of the plant or plant cell. In some instances,the mutation in the endogenous gene encoding T6ODM that was notintroduced by genetic modification of the plant or plant cell is themutation present in seeds of the plant deposited under Patent DepositDesignation PTA-9110.

Various aspects of the disclosure relate to a genetically modified poppyplant or plant cell having reduced expression of endogenous genesencoding 6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM), thegenetically modified plant comprising: a transgenic expression constructdecreasing the expression of an endogenous gene encoding T6ODM in theplant or plant cell; and a transgenic expression construct decreasingthe expression of an endogenous gene encoding CODM in the plant plant orplant cell.

Various aspects of the disclosure relate to seed of an opium poppy plantas described above.

Various aspects of the disclosure relate to use of a plant as describedabove for the production of thebaine.

Various aspects of the disclosure relate to poppy straw from a plant asdescribed above.

Various aspects of the disclosure relate to latex isolated from a plantas defined above.

Various aspects of the disclosure relate to a method of producingthebaine, said method comprising isolating thebaine from latex or poppystraw harvested from a plant as described above.

Various aspects of the disclosure relate to an isolated nucleic acidmolecule, wherein the sequence of the nucleic acid molecule comprises aportion of SEQ ID NO:7.

Various aspects of the disclosure relate to an expression vector forsimultaneously reducing the expression of endogenous genes encodingthebaine 6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM) inan opium poppy plant, the expression vector comprising a nucleic acidmolecule that comprises a portion of SEQ ID NO:7.

Various aspects of the disclosure relate to use of a portion of apolynucleotide molecule having a sequence comprising a portion of SEQ IDNO: 2 or SEQ ID NO: 4 for simultaneously reducing the expression ofendogenous genes encoding thebaine 6-O-demethylase (T6ODM) and codeine3-O-demethylase (CODM) in an opium poppy plant.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

The methods disclosed herein may be useful for producing poppy plantshaving an increased ratio of thebaine:morphine.

Particular aspects of the disclosure relate to a method of increasingaccumulation of thebaine in an opium poppy plant or plant cell, themethod comprising genetically modifying the genome of the plant or plantcell to include one or more stable genetic modifications tosimultaneously reduce the activity of thebaine 6-O-demethylase (T6ODM)and codeine 3-O-demethylase (CODM) in the poppy plant or plant cell.

Particular aspects of the disclosure relate to a genetically modifiedopium poppy plant or plant cell having reduced activity of thebaine6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM) relative to awild type plant or plant cell, wherein the genetically modified opiumpoppy plant or plant cell comprises one or more stable geneticmodifications to reduce expression of T6ODM, CODM, or both.

Particular aspects of the disclosure relate to a method for producing anopium poppy plant with increased thebaine content, the methodcomprising: (a) decreasing the expression of an endogenous gene encodingan endogenous thebaine 6-O-demethylase (T6ODM) in the plant; and (b)decreasing the expression of an endogenous gene encoding codeine3-O-demethylase (CODM) in the plant, wherein decreasing the expressionof the endogenous gene encoding T6ODM comprises genetically modifyingthe plant to have a loss of function allele in the endogenous geneencoding T6ODM.

Particular aspects of the disclosure relate to a method for producing anopium poppy plant with increased thebaine content, the methodcomprising: (a) decreasing the expression an endogenous gene encoding anendogenous thebaine 6-O-demethylase (T6ODM) in the plant; and (b)decreasing the expression of an endogenous gene encoding codeine3-O-demethylase (CODM) in the plant, wherein decreasing the expressionof the endogenous gene encoding T6ODM comprises expressing aheterologous nucleic acid molecule homologous to a portion of theendogenous gene encoding T6ODM, wherein expression of the heterologousnucleic acid molecule decreases expression of the endogenous geneencoding T6ODM.

Particular aspects of the disclosure to a method for producing an opiumpoppy plant with increased thebaine content, the method comprising: (a)decreasing the expression an endogenous gene encoding an endogenousthebaine 6-O-demethylase (T6ODM) in the plant; and (b) decreasing theexpression of an endogenous gene encoding codeine 3-O-demethylase (CODM)in the plant, wherein decreasing the expression of the endogenous geneencoding CODM comprises genetically modifying the plant to have a lossof function allele in the endogenous gene encoding CODM.

Particular aspects of the disclosure to a method for producing an opiumpoppy plant with increased thebaine content, the method comprising: (a)decreasing the expression of an endogenous gene encoding an endogenousthebaine 6-O-demethylase (T6ODM) in the plant; and (b) decreasing theexpression of an endogenous gene encoding codeine 3-O-demethylase (CODM)in the plant, wherein decreasing the expression of the endogenous geneencoding CODM comprises expressing a heterologous nucleic acid moleculehomologous to a portion of the endogenous gene encoding CODM, whereinexpression of the heterologous nucleic acid molecule decreasesexpression of the endogenous gene encoding CODM.

Particular aspects of the disclosure relate to a genetically modifiedpoppy plant or plant cell having reduced expression of endogenous genesencoding 6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM), thegenetically modified plant comprising: a stably inherited transgenicexpression construct for decreasing the expression of an endogenous geneencoding T6ODM in the plant or plant cell; and a stably inheritedtransgenic expression construct for decreasing the expression of anendogenous gene encoding CODM in the plant or plant cell.

Particular aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a first plant as comprising a loss of functionallele in an endogenous gene encoding codeine 3-O-demethylase (CODM);ii) establishing a cross of said first plant to a second plant having aloss of function allele in an endogenous gene encoding thebaine6-O-demethylase (T6ODM); iii) allowing progeny from the cross toself-fertilize; and iv) screening progeny from self-fertilized plantsfor a plant that is homozygous for both the loss of function allele inthe endogenous gene encoding CODM and the loss of function allele in theendogenous gene encoding T6ODM.

Particular aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a first plant as comprising a loss of functionallele in an endogenous gene encoding thebaine 6-O-demethylase (T6ODM);ii) establishing a cross of said first plant to a second plant having aloss of function allele in an endogenous gene encoding codeine3-O-demethylase (CODM); iii) allowing progeny from the cross toself-fertilize; and iv) screening progeny from self-fertilized plantsfor a plant that is homozygous for both the loss of function allele inthe endogenous gene encoding CODM and the loss of function allele in theendogenous gene encoding T6ODM.

Particular aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a plant as comprising a loss of function allelein an endogenous gene encoding thebaine 6-O-demethylase (T6ODM); ii)genetically modifying the plant to introduce a loss of function allelein an endogenous gene encoding codeine 3-O-demethylase (CODM); iii)allowing the plant to self-fertilize; and iv) screening progeny from theself-fertilized plant for a plant that is homozygous for both the lossof function allele in the endogenous gene encoding CODM and the loss offunction allele in the endogenous gene encoding T6ODM.

Particular aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a plant as comprising a loss of function allelein an endogenous gene encoding codeine 3-O-demethylase (CODM); ii)genetically modifying the plant to introduce a loss of function allelein an endogenous gene encoding thebaine 6-O-demethylase (T6ODM) in theplant by genetic modification; iii) allowing the plant toself-fertilize; and iv) screening progeny from the self-fertilized plantfor a plant that is homozygous for both the loss of function allele inthe endogenous gene encoding CODM and the loss of function allele in theendogenous gene encoding T6ODM.

Particular aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a plant as comprising a loss of function allelein an endogenous gene encoding thebaine 6-O-demethylase (T6ODM); ii)genetically modifying the plant to reduce expression of an endogenousgene encoding codeine 3-O-demethylase (CODM); iii) allowing the plant toself-fertilize; and iv) screening progeny from the self-fertilized plantfor a plant that is homozygous for the loss of function allele in theendogenous gene encoding T6ODM and has reduced expression of theendogenous gene encoding CODM.

Particular aspects of the disclosure relate to a method of generating anopium poppy plant having increased thebaine content relative to a wildtype opium poppy plant, the method comprising: i) using a molecularmethodology to identify a plant as comprising a loss of function allelein an endogenous gene encoding codeine 3-O-demethylase (CODM); ii)genetically modifying the plant to reduce expression of an endogenousgene encoding thebaine 6-O-demethylase (T6ODM); iii) allowing the plantto self-fertilize; and iv) screening progeny from the self-fertilizedplant for a plant that is homozygous for both the loss of functionallele in the endogenous gene encoding CODM and has reduced expressionof the endogenous gene encoding T6ODM.

Particular aspects of the disclosure relate to an isolated nucleic acidmolecule, wherein the sequence of the nucleic acid molecule comprisesSEQ ID NO:7.

Particular aspects of the disclosure relate to a use of a polynucleotidemolecule having a sequence comprising a portion of SEQ ID NO: 2 or SEQID NO: 4 for simultaneously reducing the expression of endogenous genesencoding thebaine 6-O-demethylase (T6ODM) and codeine 3-O-demethylase(CODM) in an opium poppy plant.

Particular aspects of the disclosure relate to poppy straw harvest froma plant or plant cell as claimed.

Particular aspects of the disclosure related to latex harvested from aplant or a plant cell as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate embodiments of the invention,

FIG. 1 is a schematic diagram of the morphine biosynthesis pathway inopium poppy, showing two routes from thebaine to morphine.

FIG. 2 is a schematic diagram of a hairpin RNA expression cassette forexpressing a hairpin RNA comprising T6ODM sequences for simultaneousreduction of expression of the genes encoding CODM and T6ODM.

FIG. 3 is a is an alignment of cDNA sequences encoding CODM and T6ODM.The underlined portion identifies T6ODM sequences used in the creationof the hairpin RNA expression construct to reduce expression of both theendogenous gene encoding CODM and the endogenous gene encoding T6ODM.Differences between the T6ODM and CODM coding sequences within theunderlined portion are highlighted in black.

FIG. 4 are histograms showing the expression of the endogenous genesencoding CODM and T6ODM after transient expression of the expressionconstruct.

FIG. 5 is a histogram showing expression of the expression construct intransgenic lines AM1, AM2, and AM3.

FIGS. 6A-B are histograms showing reduced expression of the endogenousgenes encoding T6ODM in FIG. 6A and CODM in FIG. 6B in lines AM1, AM2,and AM3.

FIGS. 7A-D are chromatographs showing the accumulation of thebaine inlines AM1 in FIG. 7A, AM2 in FIG. 7B, and AM3 in FIG. 7C relative toline AM10 in FIG. 7D that does not have the expression construct.

DETAILED DESCRIPTION

This disclosure relates to a genetically modified opium poppy plants,seeds, cells, straw, progeny thereof, or produced latex thereof, whichgenetically modified plant produces a latex having increased levels ofthebaine relative to wild type plants due to the combined reduction inthe activity of the enzymes thebaine 6-O-demethylase (T6ODM) and codeine3-O-demethylase (CODM) during opiate biosynthesis. The disclosure alsorelates to methods of obtaining such genetically modified opium poppyplants.

Definitions

“Opium poppy plant” or “poppy plant” as used herein refers to a plant ofthe species Papaver Somniferum.

A “field” of plants as used herein, refers to a plurality of opiumplants cultivated together in close proximity.

“Activity” or as used herein refers to the level of a particularlyenzymatic function in a plant cell. In the context of the presentdisclosure, reduced T6ODM activity refers to a reduction inO-demethylation activity at position 6, whereas reduced CODM activityrefers to a reduction in O-demethylation activity at position 3.Reduction in activity can be the result of diminished functionality ofthe protein due to, for example, mutation, or the result of reducedexpression of the protein, for example, due to reduced translation.

A “genetic modification” as used herein broadly refers to any a novelcombination of genetic material obtained with techniques of modernbiotechnology. Genetic modifications include, but are not limited to,“transgenes” in which the genetic material has been altered by theinsertion of exogenous genetic material. However, genetic modificationsalso include alterations (e.g. insertions, deletions, or substitutions)in endogenous genes introduced in a targeted manner with techniques suchas CRISPR/Cas9, TALENS, etc. as discussed below. However, for thepurposes of this disclosure “genetic modification” is not intended toinclude novel combinations of genetic material resulting from mutationsgenerated by traditional means of random mutagenesis following bytraditional means of breeding.

“Transgene” as used herein refers to a recombinant gene or geneticmaterial that has been transferred by genetic engineering techniquesinto the plant cell. “Transgenic plants” or “transformed plants” as usedherein refers to plants that have incorporated or integrated exogenousnucleic acid sequences or DNA fragments into the plant cell. A transgenemay include a homologous or heterologous promoter operably linked to aDNA molecule encoding the RNA or polypeptide of interest.

“Operably linked” refers to a functional linkage between a promoter anda second DNA sequence, wherein the promoter sequence initiates andmediates transcription of the DNA sequence corresponding to the secondDNA sequence. Generally, operably linked means that the nucleic acidsequences being linked are contiguous.

A “genetically modified” plant or plant cell as used herein broadlyrefers to any plant or plant cell that possesses a genetic modificationas defined herein.

As used herein, the term “polypeptide” encompasses any chain ofnaturally or non-naturally occurring amino acids (either D- or L-aminoacids), regardless of length (e.g., at least 5, 6, 8, 10, 12, 14, 16,18, 20, 25, 30, 40, 50, 100 or more amino acids) or post-translationalmodification (e.g., glycosylation or phosphorylation) or the presence ofe.g. one or more non-amino acyl groups (for example, sugar, lipid, etc.)covalently linked to the peptide, and includes, for example, naturalproteins, synthetic or recombinant polypeptides and peptides, hybridmolecules, peptoids, peptidomimetics, etc. As used herein, the terms“polypeptide”, “peptide” and “protein” may be used interchangeably.

“Nucleotide sequence”, “polynucleotide sequence”, “nucleic acid” or“nucleic acid molecule” as used herein refers to a polymer of DNA or RNAwhich can be single or double stranded and optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. “Nucleic acid”, “nucleic acidsequence”, “polynucleotide sequence” or “nucleic acid molecule”encompasses genes, cDNA, DNA and RNA encoded by a gene. Nucleic acids,nucleic acid sequences, polynucleotide sequence and nucleic acidmolecule may comprise at least 3, at least 10, at least 100, at least1000, at least 5000, or at least 10000 nucleotides or base pairs.

A “fragment”, a “fragment thereof”, “gene fragment” or a “gene fragmentthereof” as used herein refers to a portion of a “nucleotide sequence”,“polynucleotide sequence”, “nucleic acid” or “nucleic acid molecule”that may still reduce expression of the gene(s) encoding CODM and/orT6ODM. In one embodiment, the fragment comprises at least 20, at least40, at least 60, at least 80, at least 100, at least 150, at least 200,at least 150, at least 300, at least 350, at least 400, at least 450 orat least 500 contiguous nucleotides.

A “non-natural variant” as used herein refers to nucleic acid sequencesnative to an organism but comprising modifications to one or more of itsnucleotides introduced by mutagenesis.

An “allele” or “allelic variant” as used herein refers to an alternateform of the same gene at a specific location of the genome.

“Wildtype” as used herein refers to a plant or plant material that wasnot transformed with a nucleic acid molecule or construct, geneticallymodified, or otherwise mutated as described herein. A “wildtype” mayalso refer to a plant or plant material in which T6ODM activity and CODMactivity were not reduced.

The term “identity” as used herein refers to sequence similarity betweentwo polypeptide or polynucleotide molecules. Identity can be determinedby comparing each position in the aligned sequences. A degree ofidentity between amino acid or nucleic acid sequences is a function ofthe number of identical or matching amino acids or nucleic acids atpositions shared by the sequences, for example, over a specified region.Optimal alignment of sequences for comparisons of identity may beconducted using a variety of algorithms, as are known in the art,including the Clustal W™ program, the local homology algorithm of Smithand Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Natl.Acad. Sci. USA 85:2444, and the computerised implementations of thesealgorithms (such as GAP, BESTFIT, FASTA and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, Madison, Wis.,U.S.A.). Sequence identity may also be determined using the BLASTalgorithm (e.g. BLASTn and BLASTp), described in Altschul et al., 1990,J. Mol. Biol. 215:403-10 (using the published default settings).Software for performing BLAST analysis is available through the NationalCenter for Biotechnology Information. For instance, sequence identitybetween two nucleic acid sequences can be determined using the BLASTnalgorithm at the following default settings: expect threshold 10; wordsize 11; match/mismatch scores 2, −3; gap costs existence 5, extension2. Sequence identity between two amino acid sequences may be determinedusing the BLASTp algorithm at the following default settings: expectthreshold 10; word size 3; matrix BLOSUM 62; gap costs existence 11,extension 1. In another embodiment, the person skilled in the art canreadily and properly align any given sequence and deduce sequenceidentity/homology by mere visual inspection.

As used herein, “heterologous”, “foreign” and “exogenous” DNA and RNAare used interchangeably and refer to DNA or RNA that does not occurnaturally as part of the plant genome in which it is present or which isfound in a location or locations in the genome that differ from that inwhich it occurs in nature. Thus, heterologous or foreign DNA or RNA isnucleic acid that is not normally found in the host genome in anidentical context (i.e. linked to identical 5′ and 3′ sequences). In oneaspect, heterologous DNA may be the same as the host DNA but introducedinto a different place in the host genome and/or has been modified bymethods known in the art, where the modifications include, but are notlimited to, insertion in a vector, linked to a foreign promoter and/orother regulatory elements, or repeated at multiple copies. In anotheraspect, heterologous DNA may be from a different organism, a differentspecies, a different genus or a different kingdom, as the host DNA.Further, the heterologous DNA may be a transgene. As used herein,“transgene” refers to a segment of DNA containing a gene sequence thathas been isolated from one organism and introduced into a differentorganism. In the context of the present disclosure, the nucleic acidmolecules may comprise nucleic acid that is heterologous to the plant inwhich CODM and T6ODM activity is reduced.

“Expression” or “expressing”, as used herein refers to the process bywhich information from a gene is used in the synthesis of a functionalgene product, and may relate to production of any detectable level of aproduct, or activity of a product, encoded by a gene. Gene expressionmay be modulated (i.e. initiated, increased, decreased, terminated,maintained or precluded) at many levels including transcription, RNAprocessing, translation, post-translational modification, proteindegradation. Gene expression can also be modulated by the introductionof mutations that affect the activity of the gene product, e.g. theability of a gene product to convert substrate. In the context of thepresent disclosure, reduced expression of the endogenous gene(s)encoding CODM and/or T6ODM, or reduced expression of the CODM and/orT6ODM polypeptides, can be effected by reduced transcription of theendogenous gene(s) encoding CODM and/or T6ODM, by reduced translation ofmRNA transcripts coding for CODM and/or T6ODM, or by the introduction ofmutations that either prevent the translation of functional polypeptidesor result in the translation of polypeptides with reduced abilities toconvert substrate. Such reduced expression of the endogenous genes mayresult from expression of transgenes comprising expression constructsdesigned to reduce expression of the endogenous genes.

“Poppy straw” as used herein refers to the straw material resulting fromthreshing of mature poppy capsules and the poppy capsule stems to removethe seeds.

“Latex” as used herein refer to the air-dried, milky exudation fromlansed, unripe poppy capsules.

The term “increased thebaine content” or “increased level of thebaine”as used herein refers to a significantly increased levels of thebaine inone or more tissues as compared to the levels of thebaine in acorresponding wild type plant. The term “increased” also encompasseslevels of thebaine that are significantly increased in one or moretissues compared to the same tissues of a wild type plant, while wildtype levels of thebaine persist elsewhere in the plant.

The term “reduced morphine content” as used herein refers to asignificantly decreased levels of morphine in one or more tissues ascompared to the levels of morphine in a corresponding wild type plant.The term “reduced” also encompasses levels of morphine that aresignificantly reduced in one or more tissues compared to the sametissues of a wild type plant, while wild type levels of morphine persistelsewhere in the plant.

“Decreasing expression”, “decreasing activity”, “reducing expression”,and “reducing activity” are intended to encompass well known equivalentterms regarding expression and activity such as “inhibiting”,“down-regulating”, “knocking out”, “silencing”, etc.

“substantially no” when referring to alkaloid content means that theparticular alkaloid or combination of alkaloids constitutes less than0.6% by weight, preferably, less than 0.5% by weight, more preferably,less than 0.4% by weight, or less than 0.2% by weight of the alkaloidcombination of the poppy straw, concentrate of poppy straw or opium.

“Expression construct” as used herein refers to any type of geneticconstruct containing a nucleic acid coding for a gene product in whichpart or all of the nucleic acid encoding sequence is capable of beingtranscribed. The transcript may be translated into a protein, but itneed not be. In certain embodiments, expression includes bothtranscription of a gene and translation of mRNA into a gene product. Inother embodiments, expression only includes transcription of the nucleicacid encoding a gene of interest into, for example, an siRNA.

An expression construct of the disclosure nucleic acid molecule mayfurther comprise a promoter and other regulatory elements, for example,an enhancer, a silencer, a polyadenylation site, a transcriptionterminator, a selectable marker or a screenable marker.

As used herein, a “vector” or a “construct” may refer to any recombinantpolynucleotide molecule such as a plasmid, cosmid, virus, vector,autonomously replicating polynucleotide molecule, phage, or linear orcircular single-stranded or double-stranded DNA or RNA polynucleotidemolecule, derived from any source. A “vector” or a “construct” maycomprise a promoter, a polyadenylation site, an enhancer or silencer anda transcription terminator, in addition to a nucleotide sequenceencoding a gene or a gene fragment of interest. As used herein, a“transformation vector” may refer to a vector used in the transformationof, or in the introduction of DNA into, cells, plants or plantmaterials.

As used herein, a “promoter” refers to a nucleotide sequence thatdirects the initiation and rate of transcription of a coding sequence(reviewed in Roeder, Trends Biochem Sci, 16: 402, 1991). The promotercontains the site at which RNA polymerase binds and also contains sitesfor the binding of other regulatory elements (such as transcriptionfactors). Promoters may be naturally occurring or synthetic (see Datlaet al. Biotech Ann. Rev 3:269, 1997 for review of plant promoters).Further, promoters may be species specific (for example, active only inB. napus); tissue specific (for example, the napin, phaseolin, zein,globulin, dlec2, γ-kafirin seed specific promoters); developmentallyspecific (for example, active only during embryogenesis); constitutive(for example maize ubiquitin, rice ubiquitin, rice actin, Arabidopsisactin, sugarcane bacilliform virus, CsVMV and CaMV 35S, Arabidopsispolyubiquitin, Solanum bulbocastanum polyubiquitin, Agrobacteriumtumefaciens-derived nopaline synthase, octopine synthase, and mannopinesynthase gene promoters); or inducible (for example the stilbenesynthase promoter and promoters induced by light, heat, cold, drought,wounding, hormones, stress and chemicals). A promoter includes a minimalpromoter that is a short DNA sequence comprised of a TATA box or an lnrelement, and other sequences that serve to specify the site oftranscription initiation, to which regulatory elements are added forcontrol of expression. A promoter may also refer to a nucleotidesequence that includes a minimal promoter plus DNA elements thatregulates the expression of a coding sequence, such as enhancers andsilencers. Thus in one aspect, the expression of the constructs of thepresent disclosrue may be regulated by selecting a species specific, atissue specific, a development specific or an inducible promoter.

“Constitutive promoter” as used herein refers to a promoter which drivesthe expression of the downstream-located coding region in a plurality ofor all tissues irrespective of environmental or developmental factors.

The skilled person will understand that it would be important to use apromoter that effectively directs the expression of the construct in thetissue in which thebaine is being synthesized. For example, theendogenous T6ODM or CODM promoters could be used. Alternatively,constitutive, tissue-specific, or inducible promoters useful under theappropriate conditions to direct high level expression of the introducedexpression construct during opiod biosynthesis can be employed.

Enhancers and silencers are DNA elements that affect transcription of alinked promoter positively or negatively, respectively (reviewed inBlackwood and Kadonaga, Science, 281: 61, 1998).

Polyadenylation site refers to a DNA sequence that signals the RNAtranscription machinery to add a series of the nucleotide A at about 30bp downstream from the polyadenylation site.

Transcription terminators are DNA sequences that signal the terminationof transcription. Transcription terminators are known in the art. Thetranscription terminator may be derived from Agrobacterium tumefaciens,such as those isolated from the nopaline synthase, mannopine synthase,octopine synthase genes and other open reading frame from Ti plasmids.Other terminators may include, without limitation, those isolated fromCaMV and other DNA viruses, dlec2, zein, phaseolin, lipase, osmotin,peroxidase, PinII and ubiquitin genes, for example, from Solanumtuberosum.

In the context of the disclosure the nucleic acid construct may furthercomprise a selectable marker. Selectable markers may be used to selectfor plants or plant cells that contain the exogenous genetic material.The exogenous genetic material may include, but is not limited to, anenzyme that confers resistance to an agent such as a herbicide or anantibiotic, or a protein that reports the presence of the construct.

Numerous plant selectable marker systems are known in the art and areconsistent with this invention. The following review article illustratesthese well known systems: Miki and McHugh; Journal of Biotechnology 107:193-232; Selectable marker genes in transgenic plants: applications,alternatives and biosafety (2004).

Examples of a selectable marker include, but are not limited to, a neogene, which codes for kanamycin resistance and can be selected for usingkanamycin, NptII, G418, hpt etc.; an amp resistance gene for selectionwith the antibiotic ampicillin; an hygromycinR gene for hygromycinresistance; a BAR gene (encoding phosphinothricin acetyl transferase)which codes for bialaphos resistance including those described inWO/2008/070845; a mutant EPSP synthase gene, aadA, which encodesglyphosate resistance; a nitrilase gene, which confers resistance tobromoxynil; a mutant acetolactate synthase gene (ALS), which confersimidazolinone or sulphonylurea resistance, ALS, and a methotrexateresistant DHFR gene.

Further, screenable markers that may be used in the context of theinvention include, but are not limited to, a β-glucuronidase or uidAgene (GUS), which encodes an enzyme for which various chromogenicsubstrates are known, green fluorescent protein (GFP), and luciferase(LUX).

Alkaloid Production in Papaver somniferum

FIG. 1 is a schematic diagram depicting two routes of morphinebiosynthesis from thebaine. O-demethylation of thebaine at position 6(ring C) is catalyzed by thebaine 6-O-demethylase (T6ODM) whereasO-demethylation at position 3 (ring A) is catalyzed by codeineO-demethylase (CODM). Thus, thebaine can undergo O-demethylation atposition 6 or position 3 to yield neopinone or oripavine, respectively.Neopinone converts spontaneously to codeinone, which is then reduced tocodeine by codeinone reductase (COR). Codeine is demethylated atposition 3 by CODM to produce morphine. Demethylation of oripavine atpostion 6 by T6ODM yields morphinone, which is then reduced to morphineby COR.

The present inventor hypothesized that it may be possible to produceplants containing elevated levels of thebaine, and reduced levels ofcodeine and morphine, compared to parental plants by simultaneouslyreducing the activity of the T6ODM and CODM enzymes.

Wild type amino acid sequences of the T6ODM and CODM enzymes arepresented in SEQ ID NOs: 1 and 3, respectively. The cDNA sequencecorresponding to the endogenous gene coding for the T6ODM enzyme ispresented as SEQ ID NO:2, and the cDNA sequence corresponding to theendogenous gene coding for the CODM enzyme is presented as SEQ ID NO:4.However, the skilled person will readily understand that naturallyoccurring variations in the T6ODM and CODM genes may exist betweenvarieties, with slightly different nucleic acid sequences that encodethe same functional protein.

Reduction of CODM and T6ODM Activity or Expression

CODM and T6ODM expression and/or activity in genetically modified plantsof the present invention may be reduced by any method that results inreduced activity of these enzymes in the plant. This may be achieved bye.g. by altering CODM and T6ODM activity at the DNA, mRNA and/or proteinlevels.

As used herein, “activity” refers to the biochemical reaction of anenzyme with its cognate substrate. In the context of the invention,reduced T6ODM (or CODM) activity may result from reduced protein levelsof T6ODM (or CODM) enzyme and/or the reduced rate at which a T6ODM (orCODM) enzyme catalyzes its reaction with thebaine.

Mutating Endogenous Genes Encoding CODM and T6ODM

In one aspect, the present disclosure relates to genetic modificationstargeting the endogenous genes encoding CODM and T6ODM to alter CODM andT6ODM expression and/or activity. The endogenous CODM and T6ODM genesmay be altered by, without limitation, knocking-out CODM and T6ODMgenes; or knocking-in a heterologous DNA to disrupt CODM and T6ODMgenes. The skilled person would understand that these approaches may beapplied to the coding sequences, the promoter or other regulatoryelements necessary for gene transcription. For example, technologiessuch as CRISPR/Cas9 and TALENS can be used to introduce loss of functionmutations in both the endogenous genes encoding CODM and T6ODM. Plantshaving at least one allele of each gene comprising such loss of functionmutations can then be self-fertilized to produce progeny homozygous forthe loss of function alleles in the genes encoding CODM and T6ODM. Insome embodiments, genetic modification of the endogenous gene encodingthe CODM (or T6ODM) enzyme results in a polypeptide that differs insequence by one or more amino acid insertions, deletions, orsubstitutions, and has diminished or no CODM (or T6ODM) activity.

Deletions involve lack one or more residues of the endogenous protein.For the purposes of this disclosure, a deletion variant includesembodiments in which no amino acids of the endogenous protein aretranslated, e.g. where the initial “start” methionine is substituted ordeleted.

Insertional mutations typically involve the addition of material at anon-terminal point in the polypeptide, but may include fusion proteinscomprising amino terminal and carboxy terminal additions. Substitutionalvariants typically involve a substitution of one amino acid for anotherat one or more sites within the protein, and may be designed to modulateone or more properties of the polypeptide. Substitutions of this kindmay, in some embodiments, be conservative, i.e. where one amino acid isreplaced with one of similar shape, size, charge, hydrophobicity,hydrophilicity, etc. Conservative substitutions are well known in theart and include, for example, the changes of: alanine to serine;arginine to lysine; asparagine to glutamine or histidine; aspartate toglutamate; cysteine to serine; glutamine to asparagine; glutamate toaspartate; glycine to proline; histidine to asparagine or glutamine;isoleucine to leucine or valine; leucine to valine or isoleucine; lysineto arginine; methionine to leucine or isoleucine; phenylalanine totyrosine, leucine or methionine; serine to threonine; threonine toserine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine;and valine to isoleucine or leucine.

Accordingly, the CODM enzyme may have an amino acid sequence thatpossesses at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to SEQ ID NO: 1. Accordingly, the T6ODM enzyme mayhave an amino acid sequence that possesses at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity to SEQ ID NO: 2.

Expression of Transgenes Targeting the Endogenous Genes

In another aspect, the present disclosure relates to reducing theexpression and/or activity of CODM and T6ODM by targeting theirrespective mRNA transcripts. In this regard, levels of CODM and T6ODM TmRNA transcripts may be reduced by methods known in the art including,but not limited to, co-suppression, antisense expression, small hair pin(shRNA) expression, interfering RNA (RNAi) expression, double stranded(dsRNA) expression, inverted repeat dsRNA expression, micro interferingRNA (miRNA), simultaneous expression of sense and antisense sequences,or a combination thereof.

In one embodiment, the present disclosure relates to the use of nucleicacid molecules that are complementary, or essentially complementary, toat least a portion of the molecules set forth in SEQ ID NO:2 or SEQ IDNO:4. Nucleic acid molecules that are “complementary” are those that arecapable of base-pairing according to the standard Watson-Crickcomplementary rules. As used herein, the term “complementary sequences”means nucleic acid sequences that are substantially complementary, asmay be assessed by the same nucleotide comparison set forth above, or asdefined as being capable of hybridizing to the nucleic acid segment ofSEQ ID NO:2 or SEQ ID NO:4 under relatively stringent conditions such asthose described herein. Nucleic acid molecules may be substantiallycomplementary (or are homologues/have identity) if the two sequenceshybridize to each other under moderately stringent, or preferablystringent, conditions. Hybridization to filter-bound sequences undermoderately stringent conditions may, for example, be performed in 0.5 MNaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989,Current Protocols in Molecular Biology, Vol. 1, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).Alternatively, hybridization to filter-bound sequences under stringentconditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel,et al. (eds), 1989, supra). Hybridization conditions may be modified inaccordance with known methods depending on the sequence of interest (seeTijssen, 1993, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y.). Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point for thespecific sequence at a defined ionic strength and pH.

The phenomenon of co-suppression in plants relates to the introductionof transgenic copies of a gene resulting in reduced expression of thetransgene as well as the endogenous gene. The observed effect depends onsequence identity between the transgene and the endogenous gene.

The term “RNA interference” (RNAi) refers to well-known methods fordown-regulating or silencing expression of a naturally occurring gene ina host plant. RNAi employs a double-stranded RNA molecule or a shorthairpin RNA to change the expression of a nucleic acid sequence withwhich they share substantial or total homology. For a review, see e.g.Agrawal, N. et al (2003) Microbiol Mol Biol Rev. 67(4): 657-685. RNA isboth an initiator and target in the process. This mechanism targets RNAfrom viruses and transposons and also plays a role in regulatingdevelopment and genome maintenance. Briefly, double stranded RNA iscleaved by the enzyme dicer resulting in short fragments of 21-23 bp(siRNA). One of the two strands of each fragment is incorporated intothe RNA-induced silencing complex (RISC). The RISC associated RNA strandpairs with mRNA and induces cleavage of the mRNA. Alternatively, RISCassociated RNA strand pairs with genomic DNA resulting in epigeneticchanges that affect gene transcription. Micro RNA (miRNA) is a type ofRNA transcribed from the genome itself and works in a similar way.Similarly, shRNA may be cleaved by dicer and associate with RISCresulting in mRNA cleavage.

Specific examples of gene silencing in poppy have been reported usingRNAi approaches. In 2008, Allen et al. reported suppression of the geneencoding the morphinan pathway enzyme salutaridinol7-O-acetyltransferase (SalAT) in opium poppy. Hairpin RNA-mediatedsuppression of SalAT resulted in the accumulation of salutaridine to 23%of total alkaloids. As discussed above in the Description of RelatedArt, Allen et al. (2004) silenced codeinone reductase (COR) in opiumpoppy using a chimeric hairpin RNA construct designed to silence allmembers of the multigene COR family through RNAi.

Antisense suppression of gene expression does not involve the catalysisof mRNA degradation, but instead involves single-stranded RNA fragmentsbinding to mRNA and blocking protein translation.

Both antisense and sense suppression are mediated by silencing RNAs(sRNAs) produced from either a sense-antisense hybrid or double strandedRNA (dsRNA) generated by an RNA-dependant RNA polymerase. Majors classesor sRNAs include short-interfering RNAs (siRNAs) and microRNAs (miRNAs)which differ in their biosynthesis.

Processing of dsRNA precursors by Dicer-Like complexes yields21-nucleotide siRNAs and miRNAs guide cleavage of target transcipts fromwithin RNA-induced silencing complexes (RISC).

T6ODM and CODM expression may be suppressed using a synthetic gene(s) oran unrelated gene(s) that contain about 21 bp regions or longer of highhomology (preferably 100% homology) to the endogenous coding sequencesfor T6ODM and CODM.

See, for example, Jorgensen R A, Doetsch N, Muller A, Que Q, Gendler, Kand Napoli C A (2006) A paragenetic perspective on integration of RNAsilencing into the epigenome and in the biology of higher plants. ColdSpring Harb. Symp. Quant. Biol. 71:481-485. For a further review, seefor example, Ossowski S, Schwab R and Weigel D (2008) Gene silencing inplants using artificial microRNAs and other small RNAs. The PlantJournal 53:674-690.

Nucleic acid molecules that are substantially identical to portions ofthe endogenous coding sequences for CODM and T6ODM may also be used inthe context of the disclosure. As used herein, one nucleic acid moleculemay be “substantially identical” to another if the two molecules have atleast 60%, at least 70%, at least 80%, at least 82.5%, at least 85%, atleast 87.5%, at least 90%, at least 92.5%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% sequence identity. Thus, anucleic acid sequence comprising a nucleic acid sequence that is atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 92.5%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 or SEQID NO: 4 may be suitable for use in the context of this disclosure. Inone embodiment, the two nucleic acid molecules each comprise at least 20identical contiguous nucleotides.

Fragments of nucleic acid sequences encoding CODM or T6ODM may be used.Such fragments may have lengths of at least 20, at least 50, at least100, at least 150, at least 200, at least 300 or at least 400 contiguousnucleotides of a nucleic acid sequence encoding a CODM or T6ODM as thecase may be. Alternatively such fragments may have a minimum length ofat least 20, at least 25, at least 30, at least 35, at least 40, atleast 45, or at least 50 contiguous nucleotides and a maximum lengthless than 3000, less than 2000, less than 1750, less than 1500, lessthan 1250, less than 1000, less than 750 or less than 500 contiguousnucleotides or any combination of such minimum and maximum lengths of anucleic acid sequence encoding CODM or T6ODM as the case may be.

In one embodiment, a genetically modified opium poppy plant of thedisclosure comprises, stably integrated into its genome a first nucleicacid molecule heterologous to the plant. The first nucleic acid moleculeencodes an RNA, e.g. a hairpin RNA, for reducing expression of the CODMenzyme. The genetically modified opium poppy plant further comprises anda second nucleic acid molecule heterologous to the plant. The secondnucleic acid molecule encodes an RNA, e.g. a hairpin RNA, for reducingexpression of the T6ODM enzyme.

The first and second nucleic acid molecules may be present in a singlegenetic construct or in multiple constructs. In one embodiment, thefirst and/or second nucleic acid molecules may be arranged in the senseorientation relative to a promoter. In another embodiment, the firstand/or second nucleic acid molecules may be arranged in the anti-senseorientation relative to a promoter. In a further embodiment, a geneticconstruct may comprise at least two nucleic acid molecules in both thesense and anti-sense orientations, relative to a promoter. A geneticconstruct comprising nucleic acids in both the sense and anti-senseorientations may result in mRNA transcripts capable of forming stem-loop(hairpin) structures.

One or both of the nucleic acid molecules may be under transcriptionalcontrol of the same promoter.

In various instances, the first and second heterologous nucleic acidmolecules respectively comprise:

-   -   at least 20, at least 50, at least 100, at least 150, at least        200, at least 300 or at least 400 contiguous nucleotides of a        nucleic acid sequence possessing at least 80%, at least 90% or        100% sequence identity to the nucleic acid sequence set forth in        SEQ ID NO: 4;    -   at least 20, at least 50, at least 100, at least 150, at least        200, at least 300 or at least 400 contiguous nucleotides of a        nucleic acid sequence possessing at least 80%, at least 90% or        100% sequence identity to the nucleic acid sequence set forth in        SEQ ID NO: 2.

In various instances, the first and second nucleic acid moleculesrespectively comprise:

-   -   a nucleic acid molecule with a minimum length of at least 20, at        least 25, at least 30, at least 35, at least 40, at least 45, or        at least 50 contiguous nucleotides and a maximum length less        than 1750, less than 1500, less than 1250, less than 1000, less        than 750 or less than 500 contiguous nucleotides or any        combination of such minimum and maximum lengths of a nucleic        acid sequence possessing at least 80%, at least 90% or 100%        sequence identity to the nucleic acid sequence set forth in SEQ        ID NO:4; and    -   a nucleic acid molecule with a minimum length of at least 20, at        least 25, at least 30, at least 35, at least 40, at least 45, or        at least 50 contiguous nucleotides and a maximum length less        than 1750, less than 1500, less than 1250, less than 1000, less        than 750 or less than 500 contiguous nucleotides or any        combination of such minimum and maximum lengths of a nucleic        acid sequence possessing at least 80%, at least 90% or 100%        sequence identity to the nucleic acid sequence set forth in SEQ        ID NO: 2.

The skilled person will also appreciate that the reduction in activityof CODM and T6ODM may not be limited by the number of different nucleicacid molecules introduced into a plant or plant cell. In one embodiment,one nucleic acid molecule may target one or both endogenous genesencoding the enzymes. Accordingly, in another embodiment, a geneticallymodified opium poppy plant of the disclosure comprises, stablyintegrated into its genome a nucleic acid molecule heterologous to theplant. The nucleic acid molecule encodes a single transcript comprisingan RNA (e.g a hairpin RNA) for reducing expression of the CODM enzymeand an RNA (e.g. a hairpin RNA) for reducing expression of the T6ODMenzyme. In the working embodiment specifically exemplified in thisdisclosure, the nucleic acid molecule encodes a single transcriptcomprising a single hairpin RNA for reducing expression of both the CODMenzyme and the T6ODM enzyme.

In one aspect, a nucleic acid molecule may comprise a portion(s) of thecoding sequence for CODM (SEQ ID NO: 4); T6ODM (SEQ ID NO: 2); anallelic variant thereof; a non-natural variant thereof; a fragmentthereof; or any combination thereof.

In one embodiment, a fragment of the coding sequence for T6ODM (SEQ IDNO: 2) is suitable for the production of an expression construct codingfor an RNAi hairpin that targets expression of the endogenous codingsequences of both CODM and T6ODM. In the working embodiment specificallyexemplified in this disclosure, such fragment comprises SEQ ID NO: 7. Inthe working embodiment specifically exemplified in this disclosure, suchexpression construct comprises SEQ ID NO: 5. In the working embodimentspecifically exemplified in this disclosure, the RNAi hairpin is encodedby a nucleic acid comprising SEQ ID NO: 6.

An expression construct comprising nucleic acids in both orientationsrelative to a promoter may further comprise a spacer to separate thenucleic acid molecules in sense orientation and those in the anti-senseorientation. As used herein, a “spacer” may comprise at least 2, atleast 5, at least 10, at least 20, at least 30, at least 40, at least50, at least 75, at least 100, at least 150, or at least 200nucleotides.

The skilled person will also readily understand that although in theforegoing illustrative examples partial CODM and T6ODM coding sequenceswere suggested for constructing the CODM and T6ODM constructs, completeCODM and T6ODM coding sequences, alternative CODM and/or T6ODM codingsequences, 5′UTR and/or 3′UTR, or mutated derivatives of these sequencescan also be used. The maximum number of nucleic acid molecules that maybe used in the context of the invention may be limited only by themaximum size of the construct that may be delivered to a target plant orplant cell using a given transformation method.

In various embodiments, genetically modified plants of the presentdisclosure may further comprise a third nucleic acid moleculeheterologous to the plant. The third nucleic acid molecule is forincreasing expression of Cyp80B3 to increase the total level ofmorphinans.

Expression of Transgenes Targeting the Endogenous CODM and T6ODMPolypeptides

In a further aspect, the disclosure relates to reducing CODM and/orT6ODM activity by targeting CODM and T6ODM at the protein level. Forexample, CODM (or T6ODM) activity may be reduced by affecting thepost-translational modification of the enzyme; or by the introduction ofa heterologous protein (e.g. a mutated form of CODM or (T6ODM) may beexpressed such that it associates with the wildtype enzyme and altersits activity or outcompetes the wildtype enzyme for substrate withoutbeing able to convert the substrate; or an antibody that bindsspecifically to the CODM (or T6ODM) enzyme.

The skilled person would also appreciate that a nucleic acid moleculecomprising the sequence of a CODM or T6ODM gene promoter and/or otherregulatory elements may be used in the context of the invention. In anembodiment, a heterologous nucleic acid molecule comprising sequences ofa CODM (or T6ODM as the case may be) gene promoter and/or regulatoryelement may be used to bias the cellular machinery away from anendogenous CODM (or T6ODM as the case may be) gene promoter thusresulting in reduced CODM (or T6ODM) expression.

The size or length of the nucleic acid construct or elements thereof,are not limited to the specific embodiments described herein. Forexample, the skilled person would appreciate that the size of atransgene element may be defined instead by transgene element function;and that the promoter element may be determined instead as one that wascapable of driving transcription at a sufficient level and in thedesired tissues. Similarly, the stem loop structure formed by the mRNAtranscribed by a nucleic acid construct of the invention, may comprise anumber of gene segments which may vary in length. For example, the stemloop may comprise 3 gene segments of about 21-30 basepairs each, inaddition to a spacer, such as an intron (126 bp plus intron).

The skilled person would appreciate that the size of the gene segmentsmay be established by the sum of the element sizes combined and maydepend on the transformation method used to deliver the transgene intothe target organism. For example, each transformation method(Agrobacterium, biolistics, VIGS-based delivery systems) may be limitedto theoretical maximum transgene sizes.

Plant Transformation

The introduction of DNA into plant cells by Agrobacterium mediatedtransfer is well known to those skilled in the art. If, for example, theTi or Ri plasmids are used for the transformation of the plant cell, atleast the right border, although more often both the right and the leftborder of the T-DNA contained in the Ti or Ri plasmid must be linked tothe genes to be inserted as flanking region. If agrobacteria are usedfor the transformation, the DNA to be integrated must be cloned intospecial plasmids and specifically either into an intermediate or abinary vector. The intermediate vectors may be integrated into the Ti orRi plasmid of the agrobacteria by homologous recombination due tosequences, which are homologous to sequences in the T-DNA. This alsocontains the vir-region, which is required for T-DNA transfer.Intermediate vectors cannot replicate in agrobacteria. The intermediatevector can be transferred to Agrobacterium tumefaciens by means of ahelper plasmid (conjugation). Binary vectors are able to replicate in E.coli as well as in agrobacteria. They contain a selection marker geneand a linker or polylinker framed by the right and left T-DNA borderregion. They can be transformed directly into agrobacteria. Theagrobacterium acting as host cell should contain a plasmid carrying avir-region. The vir-region is required for the transfer of the T-DNAinto the plant cell. Additional T-DNA may be present. Such a transformedagrobacterium is used for the transformation of plant cells. The use ofT-DNA for the transformation of plant cells has been intensively studiedand has been adequately described in standard review articles andmanuals on plant transformation. Plant explants cultivated for thispurpose with Agrobacterium tumefaciens or Agrobacterium rhizogenes canbe used for the transfer of DNA into the plant cell.

Agrobacterium transformation can be used to transform opium poppy plants(Chitty et al. (Meth. Molec. Biol, 344:383-391; Chitty et al.(Functional Plant Biol, 30: 1045-1058); Facchini et al. (Plant CellRep., 27(4):719-727)). Facchini et al. (2008) disclosed A.tumefaciens-mediated genetic transformation protocol via somaticembryogenesis for the production of fertile, herbicide-resistant opiumpoppy plants. Transformation was mediated using pCAMBIA3301, atransformation vector that harbors the phosphinothricinacetyltransferase (pat) gene driven by the cauliflower mosaic virus(CaMV) 35S promoter and the β-glucuronidase (GUS) gene also driven bythe CaMV 35S promoter. Explants were co-cultivated with A. tumefaciensin the presence of 50 1M ATP and 50 1M MgCl2. Root explants pre-culturedon callus induction medium were then used for transformation.Herbicide-resistant, proliferating callus was obtained from explants ona medium containing both 2,4-dichlorophenoxyacetic acid (2,4-D) and6-benzyladenine (BA). Globular embryo genie callus was induced byremoval of the BA from the medium, and placed on a hormone-free mediumto form somatic embryos. The somatic embryos were converted to plantletsunder specific culture conditions and transferred to soil. Plants wereallowed to mature and set seed. PAT and GUS transcripts and enzymeactivities were detected in the transgenic lines tested.

Nevertheless, the present invention is not limited to any particularmethod for transforming plant cells, and the skilled person will readilyunderstand that any other suitable method of DNA transfer into plant maybe used. Methods for introducing nucleic acids into cells (also referredto herein as “transformation”) are known in the art and include, but arenot limited to: Viral methods (Clapp. Clin Perinatol, 20: 155-168, 1993;Lu et al. J Exp Med, 178: 2089-2096, 1993; Eglitis and Anderson.Biotechniques, 6: 608-614, 1988; Eglitis et al, Avd Exp Med Biol, 241:19-27, 1988); physical methods such as microinjection (Capecchi. Cell,22: 479-488, 1980), electroporation (Wong and Neumann. Biochim BiophysRes Commun, 107: 584-587, 1982; Fromm et al, Proc Natl Acad Sci USA, 82:5824-5828, 1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston andTang. Methods Cell Biol, 43: 353-365, 1994; Fynan et al. Proc Natl AcadSci USA, 90: 11478-11482, 1993); chemical methods (Graham and van derEb. Virology, 54: 536-539, 1973; Zatloukal et al. Ann NY Acad Sci, 660:136-153, 1992); and receptor mediated methods (Curiel et al. Proc NatlAcad Sci USA, 88: 8850-8854, 1991; Curiel et al. Hum Gen Ther, 3:147-154, 1992; Wagner et al. Proc Natl Acad Sci USA, 89: 6099-6103,1992).

Another method for introducing DNA into plant cells is by biolistics.This method involves the bombardment of plant cells with microscopicparticles (such as gold or tungsten particles) coated with DNA. Theparticles are rapidly accelerated, typically by gas or electricaldischarge, through the cell wall and membranes, whereby the DNA isreleased into the cell and incorporated into the genome of the cell.This method is used for transformation of many crops, including corn,wheat, barley, rice, woody tree species and others. Biolisticbombardment has been proven effective in transfecting a wide variety ofanimal tissues as well as in both eukaryotic and prokaryotic microbes,mitochondria, and microbial and plant chloroplasts (Johnston. Nature,346: 776-777, 1990; Klein et al. Bio/Technol, 10: 286-291, 1992;Pecorino and Lo. Curr Biol, 2: 30-32, 1992; Jiao et al, Bio/Technol, 11:497-502, 1993).

Another method for introducing DNA into plant cells is byelectroporation. This method involves a pulse of high voltage applied toprotoplasts/cells/tissues resulting in transient pores in the plasmamembrane which facilitates the uptake of foreign DNA. The foreign DNAenter through the holes into the cytoplasm and then to the nucleus.

Plant cells may be transformed by liposome mediated gene transfer. Thismethod refers to the use of liposomes, circular lipid molecules with anaqueous interior, to deliver nucleic acids into cells. Liposomesencapsulate DNA fragments and then adhere to the cell membranes and fusewith them to transfer DNA fragments. Thus, the DNA enters the cell andthen to the nucleus.

Other well-known methods for transforming plant cells which areconsistent with the present invention include, but are not limited to,pollen transformation (See University of Toledo 1993 U.S. Pat. No.5,177,010); Whiskers technology (See U.S. Pat. Nos. 5,464,765 and5,302,523).

The nucleic acid constructs of the present invention may be introducedinto plant protoplasts. Plant protoplasts are cells in which its cellwall is completely or partially removed using either mechanical orenzymatic means, and may be transformed with known methods including,calcium phosphate based precipitation, polyethylene glycol treatment andelectroporation (see for example Potrykus et al., Mol. Gen. Genet., 199:183, 1985; Marcotte et al., Nature, 335: 454, 1988). Polyethylene glycol(PEG) is a polymer of ethylene oxide. It is widely used as a polymericgene carrier to induce DNA uptake into plant protoplasts. PEG may beused in combination with divalent cations to precipitate DNA and effectcellular uptake. Alternatively, PEG may be complexed with otherpolymers, such as poly(ethylene imine) and poly L lysine.

A nucleic acid molecule of the present invention may also be targetedinto the genome of a plant cell by a number of methods including, butnot limited to, targeting recombination, homologous recombination andsite-specific recombination (see review Baszcynski et al. TransgenicPlants, 157: 157-178, 2003 for review of site-specific recombinationsystems in plants). Homologous recombination and gene targeting inplants (reviewed in Reiss. International Review of Cytology, 228:85-139, 2003) and mammalian cells (reviewed in Sorrell and Kolb.Biotechnology Advances, 23: 431-469, 2005) are known in the art.

As used herein, “targeted recombination” refers to integration of anucleic acid construct into a site on the genome, where the integrationis facilitated by a construct comprising sequences corresponding to thesite of integration.

Homologous recombination relies on sequence identity between a piece ofDNA that is introduced into a cell and the cell's genome. Homologousrecombination is an extremely rare event in higher eukaryotes. However,the frequency of homologous recombination may be increased withstrategies involving the introduction of DNA double-strand breaks,triplex forming oligonucleotides or adeno-associated virus.

As used herein, “site-specific recombination” refers to the enzymaticrecombination that occurs when at least two discrete DNA sequencesinteract to combine into a single nucleic acid sequence in the presenceof the enzyme. Site-specific recombination relies on enzymes such asrecombinases, transposases and integrases, which catalyse DNA strandexchange between DNA molecules that have only limited sequence homology.Mechanisms of site specific recombination are known in the art (reviewedin Grindley et al. Annu Rev Biochem, 75: 567-605, 2006). The recognitionsites of site-specific recombinases (for example Cre and att sites) areusually 30-50 bp. The pairs of sites between which the recombinationoccurs are usually identical, but there are exceptions e.g. attP andattB of A integrase (Landy. Ann Rev Biochem, 58: 913-949, 1989).

Additional methods might be selected from the resent years ofdevelopment of methods and compositions to target and cleave genomic DNAby site specific nucleases e.g. Zinc Finger Nucleases, ZFNs,Meganucleases, Transcription Activator-Like Effector Nucleases, TALENSand Clustered Regularly Interspaced Short PalindromicRepeats/CRISPR-associated nuclease (CRISPR/Cas) with an engineeredcrRNA/tracr RNA), to induce targeted mutagenesis, induce targeteddeletions of cellular DNA sequences, and facilitate targetedrecombination of an exogenous donor DNA polynucleotide within apredetermined genomic locus. Current methods for targeted insertion ofexogenous DNA typically involve co-transformation of plant tissue with adonor DNA polynucleotide containing at least one transgene and a sitespecific nuclease, e.g., ZFN, which is designed to bind and cleave aspecific genomic locus of an actively transcribed coding sequence. Thiscauses the donor DNA polynucleotide to stably insert within the cleavedgenomic locus resulting in targeted gene addition at a specified genomiclocus comprising an actively transcribed coding sequence.

As used herein the term “zinc fingers,” defines regions of amino acidsequence within a DNA binding protein binding domain whose structure isstabilized through coordination of a zinc ion.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP. Zinc finger bindingdomains can be “engineered” to bind to a predetermined nucleotidesequence. Non-limiting examples of methods for engineering zinc fingerproteins are design and selection. A designed zinc finger protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data. (U.S. Pat. No. 6,453,242; see also WO98/53058).

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit”, also referred to as a “repeat”, is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. (U.S. PatentPublication No. 2011/0301073).

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system. Briefly, a “CRISPR DNAbinding domain” is a short stranded RNA molecule that acting in concertwith the CAS enzyme can selectively recognize, bind, and cleave genomicDNA. The CRISPR/Cas system can be engineered to create a double-strandedbreak (DSB) at a desired target in a genome, and repair of the DSB canbe influenced by the use of repair inhibitors to cause an increase inerror prone repair. (Jinek et al (2012) Science 337, p. 816-821).

Zinc finger, CRISPR and TALE binding domains can be “engineered” to bindto a predetermined nucleotide sequence, for example via engineering(altering one or more amino acids) of the recognition helix region of anaturally occurring zinc finger. Similarly, TALEs can be “engineered” tobind to a predetermined nucleotide sequence, for example by engineeringof the amino acids involved in DNA binding (the repeat variablediresidue or RVD region). Therefore, engineered DNA binding proteins(zinc fingers or TALEs) are proteins that are non-naturally occurring.Non-limiting examples of methods for engineering DNA-binding proteinsare design and selection. A designed DNA binding protein is a proteinnot occurring in nature whose design/composition results principallyfrom rational criteria. Rational criteria for design include applicationof substitution rules and computerized algorithms for processinginformation in a database storing information of existing ZFP and/orTALE designs and binding data. (U.S. Pat. No. 6,453,242; see also WO98/53058; and U.S. Publication Nos. 2011/0301073).

A “selected” zinc finger protein, CRISPR or TALE is a protein not foundin nature whose production results primarily from an empirical processsuch as phage display, interaction trap or hybrid selection.

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding a T6ODM or a CODM polypeptide, resulting inreduced expression of the gene. In particular embodiments, the zincfinger protein binds to a regulatory region of a gene encoding T6ODM orCODM. In other embodiments, the zinc finger protein binds to a messengerRNA encoding a T6ODM or a CODM polypeptide and prevents its translation.Methods of selecting sites for targeting by zinc finger proteins havebeen described, for example, in U.S. Pat. No. 6,453,242, and methods forusing zinc finger proteins to inhibit the expression of genes in plantsare described, for example, in US2003/0037355, each of which is hereinincorporated by reference. Methods of selecting sites for targeting byTALE proteins have been described in e.g. Moscou M J, Bogdanove A J,2009, A simple cipher governs DNA recognition by TAL effectors. Science326:1501.

The nucleic acid molecule becomes stably integrated into the plantgenome such that it is heritable to daughter cells in order thatsuccessive generations of plant cells have reduced CODM and T6ODMexpression. This may involve the nucleic acid molecules of the presentinvention integrating, for instance integrating randomly, into the plantcell genome. Alternatively, the nucleic acid molecules of the presentinvention may remain as exogenous, self-replicating DNA that isheritable to daughter cells. As used herein, exogenous, self-replicatingDNA that is heritable to daughter cells is also considered to be “stablyintegrated into the plant genome”.

Testing for Reduction of CODM and T6ODM Activity or Expression

Disruption of endogenous genes encoding CODM and T6ODM, theirexpression, or CODM and T6ODM enzymatic activity may be confirmed bymethods known in the art of molecular biology. For example, disruptionof endogenous genes may be assessed by PCR followed by Southern blotanalysis. CODM and T6ODM mRNA levels may, for example, be measured byreal time PCR, RT-PCR, Northern blot analysis, micro-array geneanalysis, and RNAse protection. CODM and T6ODM protein levels may,without limitation, be measured by enzyme activity assays, ELISA andWestern blot analysis. CODM and T6ODM expression, or lack thereof, maybe used as a predictor of increased thebaine accumulation. CODM and/orT6ODM enzymatic activity may be assessed biochemically or functionally.

For example, CODM (and/or T6ODM) activity may be measured biochemicallyby methods known in the art including, but not limited to, the detectionof products formed by the enzyme in the presence of any number ofheterologous substrates, for example, thebaine. CODM (and/or T6ODM)activity may also be measured functionally, for example, by assessingthebaine levels in the poppy tissues.

A genetically modified opium poppy plant of the present disclosure mayresult in the reduction of CODM and/or T6ODM activity in said plant orits seed, seedling, straw, capsules, or progeny thereof, by at least57%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or 100% relative to awild type seed, seedling, straw, capsules, or progeny thereof.

Targeted Screening for Loss of Function Mutations in CODM and/or T6ODM.

This disclosure further relates to methods of generating opium poppyplants with high levels of thebaine that involve targeted screening forloss of function mutations in the endogenous genes encoding CODM and/orT6ODM and subsequent breeding of plants to combine the mutations toobtain plants homozygous for the loss of function mutations at bothloci. Opium poppy breeders have used a variety of selection techniquesin the development of improved cultivars. However, the most successfulbreeding method involving the hybridization of parents with a variety ofdifferent desired characteristics. Such approach has been usedsuccessfully to increase capsule numbers, seed and opium yield, morphinecontent, and lodging resistance.

The term “T-DNA insertion” refers to methods utilizing transfer-DNA(T-DNA) for disrupting genes via insertional mutagenesis.Down-regulating or silencing expression of the endogenous gene(s)encoding CODM and/or T6ODM in an opium poppy plant can thus be achievedby T-DNA mutagenesis, wherein the T-DNA is used to randomly inserting inthe plant genome to introduce mutations. Subsequently, plants can bescreen for T-DNA insertiosn in the genes encoding CODM and/or T6ODM byPCR, using a primer pair comprising one primer specific for the T-DNAand one primer specifi for the gene encoding CODM (or T6ODM as the casemay be), or other high-throughput technologies. For a review of T-DNA asan insertional mutagen, see e.g. Krysan, P. J. et al. (1999) Plant Cell,11: 2283-2290. Insertional mutatgenesis using transposons could also beemployed.

Mutations (including deletions, insertions, and point mutations) canalso be introduced randomly into the genome of a plant cell by variousforms of mutagenesis to produce non-natural variants. Methods formutagenesis of plant materials, including seeds, and subsequentscreening or selection for desired phenotypes are well known, asdescribed in WO2009109012. Mutagenized plants and plant cells can alsobe specifically screened for mutations in the genes encoding CODM and/orT6ODM, for example, by TILLING (Targeting Induced Local Lesions INGenomes). Loss of function mutations present in natural plantpopulations can be identified by EcoTILLING.

Once the loss of function mutations in the endogenous genes encodingCODM and T6ODM have been identified, they can be combined throughtraditional breeding processes to produce plants homozygous for the lossof function mutations at both loci. Alternatively, a loss of functionmutation identified in the endogenous gene encoding CODM can be combinedwith mutations in the endogenous gene encoding T6ODM that are introducedby genetic modification, and vice versa. Alternatively, a loss offunction mutation identified in the endogenous gene encoding CODM can becombined with genetic modification comprising an expression constructdesigned to reduce expression of T6ODM as described above, and viceversa.

Alkaloid Collection and Analysis

Opium poppy cultivation and opium harvesting traditionally involved theprocesses of manually lancing the seed capsule and collecting the latex.However, methods to extract morphine and related compounds from opiumpoppy straw circumvented the traditional technique and makes it possibleto obtain high quality seeds and pharmaceutically valuable raw materialssimultaneously. Recovering thebaine from poppy straw or latex of anopium poppy plant is well known in the art as discussed in WO2009109012.IN addition to the particular methods described below, methods ofanalyzing alkaloid extracts from opium poppy straw or latex are alsodiscussed in WO2009109012.

EXAMPLES

Referring to FIG. 2, the inventor used a single hairpin constructtargeting genes coding for CODM and T6ODM enzymes to test the hypothesisthat plants containing elevated levels of thebaine (and reduced levelsof codeine and morphine) compared to parental plants can be produced bysimultaneously reducing the activity of the T6ODM and CODM enzymes.

Referring to FIG. 3, the coding sequences for CODM and T6ODM genes havevery high level of identity. Accordingly, the inventor created a singleexpression construct to target both the endogenous gene encoding CODMand the endogenous gene encoding T6ODM from a portion of the T6ODMcoding sequence by RNAi. The portion of the T6ODM coding sequence usedfor the sense and antisense portions of the RNAi gene construct depictedin FIG. 2 is underlined in FIG. 3. A 342 base pair sense fragment wasamplified from cDNA isolated from opium poppy plant material usingprimers SEQ ID NO: 17 AAAGGCGCGCCCCTTGTCCTCAACCAAAT and SEQ ID NO: 18AAAATTTAAATTCCACTTTTAAACAAAGC). A 342 base pair antisense fragment wasamplified from cDNA isolated from opium poppy plant material usingprimers SEQ ID NO: 19 AAAACTAGTCCTTGTCCTCAACCAAAT), OPP026 (SEQ ID NO:20 AAAGGATCCTCCACTTTTAAACAAAGC). These two fragments were used to createa nucleotide molecule with these to fragments interposed by sequencesfrom β-glucuronidase.

The sequence of the nucleic acid molecule to be transcribed to produce ahairpin RNA is provided as SEQ ID NO: 6. The T6ODM sequences areunderlined, whereas the intervening “hairpin” sequence between the T6ODMsequences is derived from coding sequences for β-glucuronidase.

The complete expression construct comprising SEQ ID NO: 6 along with theCauliflower Mosaic Virus 35S promoter and transcription and translationtermination sequences from octopine synthase was cloned into a TDNAtransfer vector. The sequences between the left and right boarders ofthe vector are provided as provided as SEQ ID NO: 5. The sequence 5′ tothe first underlined region (i.e. the “sense” T6ODM-specific sequence)comprises the 35S promoter sequence, whereas the sequences 3′ to thesecond underlined region (i.e. the “antisense” T6ODM-specific sequence)comprises the transcription and translation termination sequences fromoctopine synthase.

While this expression construct was generated using a combination oftraditional polymerase chain reaction (PCR) and cloning techniques (e.g.with restriction enzymes and ligations), the skilled person willunderstand that various conventional techniques could be used to producethe construct, including overlap extension PCR cloning or directsynthesis.

The sequence of the entire T-DNA comprising SEQ ID NO:6, from RightBorder to Left Border, is provided as SEQ ID NO: 4.

Virus-induced gene silencing (VIGS) was used to transiently test theability of the gene cassette to silence the endogenous genes encodingT6ODM and CODM. VIGS is a plant RNA-silencing technique that uses viralvectors carrying a fragment of a gene of interest to generatedouble-stranded RNA, which initiates the silencing of the target gene.pTRV1 (helper plasmid) and pTRV2 (binary vector) TRV-based VIGS vectorsto express the expression construct. Tissues were taken in 48 hrs, 72hrs, 5 day, 7 days, and 2 weeks after infiltration. As indicated inFIGS. 4a and 4b , transient expression of the expression constructresulted in substantial downregulation of transcripts from theendogenous genes encoding CODM and T6ODM at 48 h post transformation(first bar from the right in both FIGS. 3a and 3b above).

Plants stably transformed with the expression construct were thengenerated according to the following protocol.

Transformation Protocol Transformation of Poppy Hypocotyls/Roots

Media required:

LB

Agrobacterium Suspension Medium

B5 salts and vitamins containing 20 g/l sucrose, pH −5.6-5.8±0.2.

Shoot Germination Medium

Half strength Murashige and Skoog basal medium supplemented with 20 g/lsucrose, 8 g/l agar. pH −5.8±0.2.

Primary Callus Induction Medium

B5 medium containing 30 g/l sucrose, 2.0 mg/l NAA, and 0.1 mg/l BAP, 8g/l agar.

Somatic Embryo Induction Medium

B5 medium containing 1.0 mg/l NAA, 0.5 mg/l BAP, 50 mg/l paromomycin,300 mg/l timentin and 8 g/l agar.

Embryo Induction Medium

Murashige and Skoog basal medium supplemented with 30 g/l sucrose, 0.25g/l MES, 0.2 g/l myo-inositol, 1 mg/l 2,4-D, 2.5 mg/l AgNO₃, 8 g/l agar.pH −5.6±0.2. (Plus antibiotic—Timentin 300 mg/l)

Embryo Maturation and Germination

Murashige and Skoog basal medium supplemented with 30 g/l sucrose, 0.25g/l MES, 0.2 g/l myo-inositol, 1 mg/l Benzyl adenine, 1 mg/l Zeatin, 2.5mg/l AgNO₃, 8 g/l agar. pH −5.6±0.2. (Plus antibiotic—Timentin 300 mg/l)

Phytohormone-Free Plant Regeneration Medium

B5 medium containing 50 mg/L paromomycin, 300 mg/L timentin, and 8 g/Lagar

Shoot Elongation Medium

Murashige and Skoog basal medium supplemented with 30 g/l sucrose, 0.25g/l MES, 0.2 g/l myo-inositol, 0.5 mg/l Benzyl adenine, 2.5 mg/l AgNO₃,8 g/l agar. pH −5.6±0.2. (Plus antibiotic—Timentin 300 mg/l)

Rooting Medium

Murashige and Skoog basal medium supplemented with 30 g/l sucrose, 0.25g/l MES, 0.2 g/l myo-inositol, 2.5 mg/l AgNO₃, 8 g/l agar. pH −5.6±0.2.(Plus antibiotic—Timentin 300 mg/l)

Seed Sterilization and Germination

The seeds were surface-sterilized with 70% (vv⁻¹) ethanol for 30 s and1% (vv⁻¹) sodium hypochlorite solution for 2 min each three times, thenrinsed five times in sterile water. Approximately 50 seeds were placedon 25 ml of agar-solidified culture medium in jars. The basal mediumconsisted of ½ Murashige and Skoog basal medium supplemented with 30 g/lsucrose (Gamborg et al. 1968) and solidified with 0.8% (wv-¹) agar. Themedium was adjusted to pH 5.6-5.8 before adding the agar, and thensterilized by autoclaving. The seeds were germinated in a growth chamberat 25° C. under standard cool white fluorescent tubes and a 16-hphotoperiod.

Preparation of Agrobacterium tumefaciens

The binary vector pORE::ALM-MIRMAC was mobilized by electroporation inAgrobacterium tumefaciens strain EHA105. A. tumefaciens cultures weregrown at 28° C. on a gyratory shaker at 180 rpm in liquid Luria-Bertanimedium [1% tryptone, 0.5% yeast extract, and 1% NaCl, pH 7.0] containing50 mg/l kanamycin and rifampicin 100 mg/l, to A₆₀₀=0.8. The bacterialcells were collected by centrifugation for 10 min at 4000 rpm andresuspended at a cell density of A₆₀₀=0.5 in liquid inoculation medium(B5 salts and vitamins containing 20 g/l sucrose).

Production of Transgenic Plants

Excised cotyledons from 12-day-old seedlings, line 118, were isolated bylongitudinal bisection of the hypocotyl. The hypocotyls were dipped intothe A. tumefaciens culture in liquid inoculation medium for 15 min,blotted dry on sterile filter paper, and incubated in the dark at 25° C.on primary callus induction medium. After 2 days of co-cultivation withA. tumefaciens, the hypocotyls were transferred to fresh primary callusinduction medium containing 50 mg/L paromomycin and 300 mg/L timentin.After 4-5 weeks of incubation, primary calli were subcultured on somaticembryo induction medium. After 3 weeks of cultivation on inductionmedium, somatic embryos were transferred to phytohormone-free plantregeneration medium. Mature embryos were placed on phytohormone-freemedium and immature embryos were transferred to embryo maturation andgermination medium. Then, when first cotyledons appeared they weretransplanted onto shoot elongation medium. Finally, when shoots wereabout 0.5-1 cm, they were placed on rooting medium. Regenerated putativetransgenic plantlets were grown in a growth chamber at 25° C. understandard cool-white conditions and a 16-h photoperiod. Rooted plantletswere then transferred to pots containing autoclaved soil, covered withpolyethylene bags for 1 week to sustain high humidity, and maintained inthe growth chamber at 25° C. for 1-2 weeks before the plants weretransferred to the greenhouse.

Method:

-   -   1. Pick a single colony of the desired construct in        Agrobacterium and inoculate it in 2 ml LB liquid medium        containing appropriate antibiotics; culture overnight at 28° C.        to prepare a starter culture.    -   2. Inoculate 100 ml LB liquid medium containing appropriate        antibiotics with the starter culture and incubate overnight at        28° C. Incubate cells until a desired OD₆₀₀=0.5-0.8 is attained.    -   3. Pellet the cells by centrifugation at 4000 rpm for 10 mins.    -   4. Resuspend the cells in Agrobacterium suspension medium to a        final OD₆₀₀=0.5.    -   5. Roots and hypocotyl segments from 12 day old seedlings were        cut into ˜5 mm segments while dipping in the Agrobacterium        suspension.    -   6. Incubate the roots and hypocotyl segments on Petri dish for        15 mins in the Agrobacterium suspension with occasional        swirling.    -   7. Blot dry the roots and hypocotyl segments on a sterile filter        paper and transfer to plates containing primary callus induction        medium. Incubate the plates for 2 days at 22±2° C. in a growth        cabinet under dark (covered with aluminium foil).    -   8. After 2 days of co-cultivation, wash the root and hypocotyl        segments with sterile distilled water, blot dry on a sterile        filter paper and transfer to plates containing primary callus        induction medium (antibiotic paromomycin was added).    -   9. Incubate the plates in growth chamber at standard conditions        (16/8 h photoperiod) in dark (covered with aluminium foil) for        approximately 4-5 weeks.    -   10. After 4-5 weeks of incubation, primary calli were        subcultured on somatic embryo induction medium (antibiotic        paromomycin was added).    -   11. After 3 weeks of cultivation on induction medium, mature        somatic embryos were transferred to phytohormone free medium (no        antibiotic). Immature embryos were placed to another round of        selection on embryo maturation and germination medium (no        antibiotic) and incubated at 22±2° C. in a growth cabinet under        16/8 h photoperiod until embryos matured and started        germinating.    -   12. Transfer the germinating embryos to plates containing shoot        elongation medium until shoots appear and subsequently transfer        to rooting medium.    -   13. Transfer ˜0.5-1.0 cm shoots to rooting medium.    -   14. Rooted plantlets are washed under tap water to remove the        entire adhering agar and then are transferred to sterile soil in        small pots, covered with saran wrap and incubated in a growth        cabinet for acclimatization.    -   15. Obtained putative transformants are analysed for transgene        presence and expression.        -   Notes:—no antibiotic in rooting media.

Chemicals

-   -   1. 50 mg/ml Paromomycin sulfate stock: Prepare by dissolving        powder in water and sterilize by filtration, aliquot, and store        at −20 C.    -   2. 50 mg/ml Kanamycin stock: Prepare by dissolving powder in        water and sterilize by filtration, aliquot, and store at −20 C.    -   3. 100 mg/ml Rifampicin stock: Prepare by dissolving powder in        DMSO, aliquot, and store at −20 C.    -   4. 300 mg/ml Timentin stock: Prepare by dissolving powder in        water and sterilize by filtration, aliquot, and store at −20 C.    -   5. 2.0 mg/ml NAA stock: Prepare by dissolving powder in 1N NaOH,        adjust volume with water and sterilize by filtration, aliquot,        and store at −20 C.    -   6. 2.0 mg/ml BAP stock: Prepare by dissolving powder in 1N NaOH,        adjust volume with water and sterilize by filtration, aliquot,        and store at −20 C.    -   7. 5 mg/ml AgNO3 stock: Prepare by dissolving powder in water        and sterilize by filtration, and store at +4 C.    -   8. 1.0 mg/ml Zeatin stock: Prepare by dissolving powder in 1N        NaOH, adjust volume with water and sterilize by filtration,        aliquot, and store at −20 C.    -   9. All the chemicals used for media are added to autoclaved        medium once it has cooled to about 50 C. Swirl to mix thoroughly        the medium before pouring into 90×25-mm Petri dishes.

Characterization of Regenerated Transgenic Plants

The T-DNA comprising the expression construct included the nptll gene,which confers resistance to paromycin. Six plantlets (AM1 to AM6) wereidentified as resistant to paromomycin, suggesting that these plantswere transformed with the expression construct. Polymerase chainreaction on genomic DNA isolated from these plantlets using primersspecific for the hairpin expression construct (SEQ ID NO: 9,TAACCGACTTGCTGCCCCGA; SEQ ID NO: 10, AAATAGAGATGCTTGCAGAAGATCCCG) showedthat plants AM1, AM2 and AM3 contain amplicon from genomic DNA. Primersfor actin (SEQ ID NO: 11, CGTTTGAATCTTGCTGGCCGTGAT; SEQ ID NO: 12,TAGACGAGCTGCCTTTGGAAGTGT) were used as a positive control to confirmthat the samples contained genomic DNA from Papaver somniferum.

Referring to FIG. 4 reverse transcription polymerase chain reaction(RT-PCR) was performed on RNA extracts from plants AM1 to AM6 wasperformed using primers specific for the hairpin (SEQ ID NO: 9,TAACCGACTTGCTGCCCCGA; SEQ ID NO: 10, AAATAGAGATGCTTGCAGAAGATCCCG), anddemonstrated that the expression construct was expressed in plants AM1,AM2, and AM3.

RT-PCR was performed on the RNA extracts from plants AM1 to AM6 usingprimers specific for endogenous transcripts encoding CODM and T6ODM.Referring to FIG. 5, expression of the expression construct appearedsufficient to downregulate the expression of the endogenous geneencoding T6ODM relative to plants that did not express the expressionconstruct. Referring to FIG. 6, expression of the expression constructappeared sufficient to downregulate the expression of the endogenousgene encoding CODM relative to plants that did not express theexpression construct.

Alkaloid Analysis

Acidic extraction: 0.100 g of ground capsule or stems (from poppy) wasmixed with 5 ml of a solution of 10% acetic acid, 10% water and 80%methanol followed by agitation for 30 minutes, and then filtered. Thefiltrate was directly injected into the HPLC.

All samples were run on an HPLC Gradient System having an Kinetex 2.6 umC18, 50×2.1 mm column, with a 2 microliter injection volume, operatingat 280 nm at a temperature of 45° C. and a flow rate of 0.8 ml/min.Eluent A was 10 mM Ammonium acetate Buffer, pH 5.5, whereas Eluent B wasAcetonitrile (100%). The gradient profile is as follows

Step No. Time (min) Pct A Pct B 1 0 95 5 2 0.25 85 15 3 2.00 60 40 42.01 95 5 5 3.00 20 80 6 4.00 10 90 7 5.00 0 100

The reported figures are percent by weight of the dry starting material.

Referring to FIGS. 7A to 7D, analysis of alkaloids in the capsule of AM1 and the leaves of AM2 and AM3 indicated increased accumulation ofthebaine and decreased accumulation of morphine compared to a plant inwhich the expression construct was not present and had a wild type levelof T6ODM and CODM expression. Subsequent analysis of alkaloids in thecapsules of the AM2 and AM3 plants showed that thebaine accumulated to4.82% of alkaloids in the AM2 capsules and to 3.13% of total alkaloidsin the AM3 capsules. Substantially no morphine was detected in AM1, AM2,and AM3 plants.

TABLE 1 Concentration of alkaloids in transoenic plants per 100 mg drycapsule. Codeine (Conc. wt %) Thebaine (Conc. wt %) AM1 0.23 3.87 AM20.11 4.82 AM3 0.12 3.13

The progeny of the self fertlized AM1, AM2, and AM3 transformants appearto segregate 3:1, indicating that the transgenes are stably integratedand inherited.

Six additional individual transformants that carry the expressionconstruct, and in which the endogenous CODM and T6ODM transcripts couldnot be detected, were isolated (AM12, AM13, AM16, AM17, and AM19).Analysis in the capsules of these additional plants showed that thebaineaccumulated to as much as 8.28% of alkaloids in the capsules (see Table2).

TABLE 2 Concentration of thebaine in transgenic plants per 100 mg drycapsule. Thebaine (Conc. wt %) AM12 5.87 AM13 6.20 AM16 8.28 AM17 7.18AM19 4.95

Operation

While specific embodiments of the invention have been described andillustrated, such embodiments should be considered illustrative of theinvention only and not as limiting the invention as construed inaccordance with the accompanying claims.

This description contains a sequence listing in electronic form in ASCIItext format. A copy of the sequence listing is available from theCanadian Intellectual Property Office. The sequences in the sequencelisting are reproduced in the following Table.

Sequence Table SEQ ID NO: 1 (T6ODM)Met Glu Lys Ala Lys Leu Met Lys Leu Gly Asn Gly Met Glu Ile Pro1               5                   10                  15Ser Val Gln Glu Leu Ala Lys Leu Thr Leu Ala Glu Ile Pro Ser Arg            20                  25                  30Tyr Val Cys Ala Asn Glu Asn Leu Leu Leu Pro Met Gly Ala Ser Val       35                   40                  45Ile Asn Asp His Glu Thr Ile Pro Val Ile Asp Ile Glu Asn Leu Leu    50                  55                  60Ser Pro Glu Pro Ile Ile Gly Lys Leu Glu Leu Asp Arg Leu His Phe65                  70                  75                  80Ala Cys Lys Glu Trp Gly Phe Phe Gln Val Val Asn His Gly Val Asp                85                  90                  95Ala Ser Leu Val Asp Ser Val Lys Ser Glu Ile Gln Gly Phe Phe Asn            100                 105                 110Leu Ser Met Asp Glu Lys Thr Lys Tyr Glu Gln Glu Asp Gly Asp Val       115                 120                 125Glu Gly Phe Gly Gln Gly Phe Ile Glu Ser Glu Asp Gln Thr Leu Asp    130                 135                 140Trp Ala Asp Ile Phe Met Met Phe Thr Leu Pro Leu His Leu Arg Lys145                 150                 155                 160Pro His Leu Phe Ser Lys Leu Pro Val Pro Leu Arg Glu Thr Ile Glu                165                 170                 175Ser Tyr Ser Ser Glu Met Lys Lys Leu Ser Met Val Leu Phe Asn Lys            180                 185                 190Met Glu Lys Ala Leu Gln Val Gln Ala Ala Glu Ile Lys Gly Met Ser        195                 200                 205Glu Val Phe Ile Asp Gly Thr Gln Ala Met Arg Met Asn Tyr Tyr Pro    210                 215                 220Pro Cys Pro Gln Pro Asn Leu Ala Ile Gly Leu Thr Ser His Ser Asp225                 230                 235                 240Phe Gly Gly Leu Thr Ile Leu Leu Gln Ile Asn Glu Val Glu Gly Leu                245                 250                 255Gln Ile Lys Arg Glu Gly Thr Trp Ile Ser Val Lys Pro Leu Pro Asn            260                 265                 270Ala Phe Val Val Asn Val Gly Asp Ile Leu Glu Ile Met Thr Asn Gly        275                 280                 285Ile Tyr His Ser Val Asp His Arg Ala Val Val Asn Ser Thr Asn Glu    290                 295                 300Arg Leu Ser Ile Ala Thr Phe His Asp Pro Ser Leu Glu Ser Val Ile305                 310                 315                 320Gly Pro Ile Ser Ser Leu Ile Thr Pro Glu Thr Pro Ala Leu Phe Lys                325                 330                 335Ser Gly Ser Thr Tyr Gly Asp Leu Val Glu Glu Cys Lys Thr Arg Lys            340                 345                 350Leu Asp Gly Lys Ser Phe Leu Asp Ser Met Arg Ile        355                 360 SEQ ID NO: 2 (T6ODM)gttcttaatt cattaattaa tttagaaaaa tcatggagaa agcaaaactt atgaagctag   60gtaatggtat ggaaatacca agtgttcaag aattggctaa actcacgctt gccgaaattc  120catctcgata cgtatgcgcc aatgaaaacc ttttgttgcc tatgggtgca tctgtcataa  180atgatcatga aaccattcct gtcatcgata tagaaaattt attatctcca gaaccaataa  240tcggaaagtt agaattagat aggcttcatt ttgcttgcaa agaatggggt ttttttcagg  300tagtgaacca tggagtcgac gcttcattgg tggatagtgt aaaatcagaa attcaaggtt  360tctttaacct ttctatggat gagaaaacta aatatgaaca ggaagatgga gatgtggaag  420gatttggaca aggctttatt gaatcagagg accaaacact tgattgggca gatatattta  480tgatgttcac tcttccactc catttaagga agcctcactt attttcaaaa ctcccagtgc  540ctctcaggga gacaatcgaa tcctactcat cagaaatgaa aaagttatcc atggttctct  600ttaataagat ggaaaaagct ctacaagtac aagcagccga gattaagggt atgtcagagg  660tgtttataga tgggacacaa gcaatgagga tgaactatta tcccccttgt cctcaaccaa  720atctcgccat cggtcttacg tcgcactcgg attttggcgg tttgacaatc ctccttcaaa  780tcaacgaagt ggaaggatta cagataaaaa gagaggggac atggatttca gtcaaacctc  840tacctaatgc gttcgtagtg aatgttggag atattttgga gataatgact aatggaattt  900accatagtgt cgatcaccgg gcagtagtaa actcaacaaa tgagaggctc tcaatcgcaa  960catttcatga ccctagtcta gagtcggtaa taggcccaat atcaagcttg attactccag 1020agacacctgc tttgtttaaa agtggatcta catatgggga tcttgtggag gaatgtaaaa 1080caaggaagct cgatggaaaa tcatttcttg actccatgag gatttgaaaa ctcaagaaaa 1140aataatacga cgtgattgca tgtcagattc aactatcctt ttgtcgtttt ttggtgctcg 1200agtccttaat tgttttgatc attgcttttg attctaatta ataagacttt tctcaagaac 1260cacatgtaat gtacctttac tttcagaaaa taaaaagtat tgaggcacaa atgagaaaat 1320tgagagagtg cttgagaagt gtaatttctc gaaagtgcgt tgtgtttgaa aaaaaaaaaa 1380aaaaaa                                                            1386SEQ ID NO: 3 (CODM)Met Glu Thr Pro Ile Leu Ile Lys Leu Gly Asn Gly Leu Ser Ile Pro1               5                   10                  15Ser Val Gln Glu Leu Ala Lys Leu Thr Leu Ala Glu Ile Pro Ser Arg            20                   25                   30Tyr Thr Cys Thr Gly Glu Ser Pro Leu Asn Asn Ile Gly Ala Ser Val        35                   40                   45Thr Asp Asp Glu Thr Val Pro Val Ile Asp Leu Gln Asn Leu Leu Ser    50                   55                   60Pro Glu Pro Val Val Gly Lys Leu Glu Leu Asp Lys Leu His Ser Ala65                   70                 75                  80Cys Lys Glu Trp Gly Phe Phe Gln Leu Val Asn His Gly Val Asp Ala                85                   90                   95Leu Leu Met Asp Asn Ile Lys Ser Glu Ile Lys Gly Phe Phe Asn Leu            100                 105                 110Pro Met Asn Glu Lys Thr Lys Tyr Gly Gln Gln Asp Gly Asp Phe Glu        115                 120                 125Gly Phe Gly Gln Pro Tyr Ile Glu Ser Glu Asp Gln Arg Leu Asp Trp    130                 135                 140Thr Glu Val Phe Ser Met Leu Ser Leu Pro Leu His Leu Arg Lys Pro145                 150                 155                 160His Leu Phe Pro Glu Leu Pro Leu Pro Phe Arg Glu Thr Leu Glu Ser                165                 170                 175Tyr Leu Ser Lys Met Lys Lys Leu Ser Thr Val Val Phe Glu Met Leu            180                 185                 190Glu Lys Ser Leu Gln Leu Val Glu Ile Lys Gly Met Thr Asp Leu Phe       195                 200                 205Glu Asp Gly Leu Gln Thr Met Arg Met Asn Tyr Tyr Pro Pro Cys Pro    210                 215                 220Arg Pro Glu Leu Val Leu Gly Leu Thr Ser His Ser Asp Phe Ser Gly225                 230                 235                 240Leu Thr Ile Leu Leu Gln Leu Asn Glu Val Glu Gly Leu Gln Ile Arg                245                 250                 255Lys Glu Glu Arg Trp Ile Ser Ile Lys Pro Leu Pro Asp Ala Phe Ile            260                 265                 270Val Asn Val Gly Asp Ile Leu Glu Ile Met Thr Asn Gly Ile Tyr Arg        275                 280                 285Ser Val Glu His Arg Ala Val Val Asn Ser Thr Lys Glu Arg Leu Ser    290                 295                 300Ile Ala Thr Phe His Asp Ser Lys Leu Glu Ser Glu Ile Gly Pro Ile305                 310                 315                 320Ser Ser Leu Val Thr Pro Glu Thr Pro Ala Leu Phe Lys Arg Gly Arg                325                 330                 335Tyr Glu Asp Ile Leu Lys Glu Asn Leu Ser Arg Lys Leu Asp Gly Lys            340                 345                 350Ser Phe Leu Asp Tyr Met Arg Met         355                 360SEQ ID NO: 4 (CODM)gtaaagattg atatatgatc tgaagatctg acaagaaagt tcatcaaata tagagttcat   60ggagacacca atacttatca agctaggcaa tggtttgtca ataccaagtg ttcaggaatt  120ggctaaactc acgcttgcag aaattccatc tcgatacaca tgcaccggtg aaagcccgtt  180gaataatatt ggtgcgtctg taacagatga tgaaacagtt cctgtcatcg atttgcaaaa  240tttactatct ccagaacccg tagttggaaa gttagaattg gataagcttc attctgcttg  300caaagaatgg ggtttctttc agctggttaa ccatggagtc gacgctttac tgatggacaa  360tataaaatca gaaattaaag gtttctttaa ccttccaatg aatgagaaaa ctaaatacgg  420acagcaagat ggagattttg aaggatttgg acaaccctat attgaatcgg aggaccaaag  480acttgattgg actgaagtgt ttagcatgtt aagtcttcct ctccatttaa ggaagcctca  540tttgtttcca gaactccctc tgcctttcag ggagacactg gaatcctacc tatcaaaaat  600gaaaaaacta tcaacggttg tctttgagat gttggaaaaa tctctacaat tagttgagat  660taaaggtatg acagacttat ttgaagatgg gttgcaaaca atgaggatga actattatcc  720tccttgtcct cgaccagagc ttgtacttgg tcttacgtca cactcggatt ttagcggttt  780gacaattctc cttcaactta atgaagttga aggattacaa ataagaaaag aagagaggtg  840gatttcaatc aaacctctac ctgatgcgtt catagtgaat gttggagaca ttttggagat  900aatgactaat gggatttacc gtagcgtcga gcaccgggca gtagtaaact caacaaagga  960gaggctctca atcgcgacat ttcatgactc taaactagag tcagaaatag gcccaatttc 1020gagcttggtc acaccagaga cacctgcttt gttcaaaaga ggtaggtatg aggatatttt 1080gaaggaaaat ctttcaagga agcttgatgg aaaatcattt ctcgactaca tgaggatgtg 1140agaaagtgtg aacatatatt atactccaca ttgtgtttaa tatatgatga aataagttgc 1200ttttgaagta tgatgaaata agttggtttt gaagaattca tattgtgctt aaatttcgtg 1260gatgactgag agatttatta tgtaataata atgtattggt ttgaagattc tcgtctcact 1320atatgtaaga ctctgtttgg gtcaagtgat gtaatcacgg ttgaaataag ttgcttttga 1380agaattcata tggtgcttaa tattatgtaa taaataatgt attggattga aaaaaaaaaa 1440aaaaaaaaaa aa                                                     1452SEQ ID NO: 5tcctgtggttggcatgcacatacaaatggacgaacggataaaccttttcacgcccttttaaatatccgattattctaataaacgctcttttctcttaggtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacgacaatctgctagtggatctcccagtcacgacgttgtaaaacgggcgccccgcggaaagcttgctagccaattggggcccaacgttctcgagaacgtggatacttggcagtggttacttggcttttcctttattttcttttggacggaagcggtggttactttgtcacacatttaaaaaaacacgtgtttctcacttttttctattcccgtcacaaacaattttaagaaagatccatctatcgtgatctttctatcaaacaaaagaaaaaaggtcttcatagtaacgctacaacatcaaatatgtggttgctctgacatcagtcgggaaaataaggatatggcggcattggccacatctattggggtcccaacttcctttcacaaaaaaattaaattgggtgtcccaacttttatctttgatatagtgacatgagtatcgggagcattggacaatggataaaatgagaactaaaaaaattctggttaatttttgatcattgttatttaaaaggttattttatctataatctacccatattgatcagttttatttaaatttgtttagctaccgctccacgagagagatcctcatcttaaaaatggaatatggaaattacacacgaccccaaaagtatattttttctctggagaatgctatttagagctttgactatatggtctgaattagaaagacgggaaataaaatctgctaagtgatataagctctaagtaggcgatgtgtgatggagaacaccttttctttaacagtcttcatgttttacagattcgcgaacttcgaatatccctatacggtctgtctaaccctcgtgtgtcttttgagtccaagataaaggccattattgagtaacatagacatgctggaatccaaccattgaagtcacaactgtccatgtagattctttggagaatctgaaaagtcttaataaaggtggtgtttcaaagaaaacaaaacaaatgagttaagaaaaaaaaatatcatgtagtggtcgagtattatgttatttattgtgtagctaccaatctttattctttaaatctgacataaaatgctacaaactttttacctcgtctatagccccaaaaaacctaaccacggttctaaaaccacacacagtgattttggttgacgacaatgcctctccttcctcaaaacgatttatttacattttttaaatcaaatgttacattttataccataattaagtctttttacagaatacttagatggaagagatgtataaaaaaggaggaaattgtaaaaaacatatttcgatcaattaaaccaggattcataaaaatataagtatatatataaatgatgtttcgtttagcgatgaacttcactcatatgataatacttaacaatataagtacataaaaaataaaataaaattaattgtttacgaaaagtctacaaatactgcatgtataattaatgttctctttatttatttatttataccttaccaagatatatctataaccgcatagaaatagaaggcgaagagataatttccaaaaacaagaaaaacctctaagctcaaaagtctagaaggccttggatccacccatggaggttgtcacagtatcacttgtagcagttgtgatcactactttcttatacttaatcttcagagattcaagtcctaaaggtttgccaccaggtccaaaaccctggccaatagttggaaaccttcttcaacttggtgagaaacctcattctcagtttgctcagcttgctgaaacctatggtgatctcttttcactgaaactaggaagtgaaacggttgttgtagcttcaactccattagcagctagcgagattctaaagacgcatgatcgtgttctctctggtcgatacgtgtttcaaagtttccgggtaaaggaacatgtggagaactctattgtgtggtctgaatgtaatgaaacatggaagaaactgcggaaagtttgtagaacggaactttttacgcagaagatgattgaaagtcaagctgaagttagagaaagtaaggctatggaaatggtggagtatttgaagaaaaatgtaggaaatgaagtgaaaattgctgaagttgtatttgggacgttggtgaatatattcggtaacttgatattttcacaaaatattttcaagttgggtgatgaaagtagtggaagtgtagaaatgaaagaacatctatggagaatgctggaattggggaactcgacaaatccagctgattattttccatttttgggtaaattcgatttgtttggacaaagaaaagatgttgctgattgtctgcaagggatttatagtgtttggggtgctatgctcaaagaaagaaaaatagccaagcagcataacaacagcaagaagaatgattttgttgagattttgctcgattccggactcgatgaccagcagattaatgccttgctcatggaaatatttggtgcgggaacagagacaagtgcatctacaatagaatgggcgttgtctgagctcacaaaaaaccctcaagtaacagccaatatgcggttggaattgttatctgtggtagggaagaggccggttaaggaatccgacataccaaacatgccttatcttcaagcttttgttaaagaaactctacggcttcatccagcaactcctctgctgcttccacgtcgagcacttgagacctgcaaagttttgaactatacgatcccgaaagagtgtcagattatggtgaacgcctggggcattggtcgggatccaaaaaggtggactgatccattgaagttttcaccagagaggttcttgaattcgagcattgatttcaaagggaacgacttcgagttgataccatttggtgcagggagaaggatatgtcctggtgtgcccttggcaactcaatttattagtcttattgtgtctagtttggtacagaattttgattggggattaccgaagggaatggatcctagccaactgatcatggaagagaaatttgggtgacactgcaaaaggaaccacctctgtatattgttcctaaaactcgggattaagggagaattcgtcgactttgcggccgcatcgatactgcaggagctcggtaccttttactagtgatatccctgtgtgaaattgttatccgctacgcgtgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcccatgggaagttcctattccgaagttcctattctctgaaaagtataggaacttcagcgatcgctccaatcccacaaaaatctgagcttaacagcacagttgctcctctcagagcagaatcgggtattcaacaccctcatatcaactactacgttgtgtataacggtccacatgccggtatatacgatgactggggttgtacaaaggcggcaacaaacggcgttcccggagttgcacacaagaaatttgccactattacagaggcaagagcagcagctgacgcgtacacaacaagtcagcaaacagacaggttgaacttcatccccaaaggagaagctcaactcaagcccaagagctttgctaaggccctaacaagcccaccaaagcaaaaagcccactggctcacgctaggaaccaaaaggcccagcagtgatccagccccaaaagagactcctttgccccggagattacaatggacgatttcctctatctttacgatctaggaaggaagttcgaaggtgaaggtgacgacactatgttcaccactgataatgagaaggttagcctcttcaatttcagaaagaatgctgacccacagatggttagagaggcctacgcagcaggtctcatcaagacgatctacccgagtaacaatctccaggagatcaaataccttcccaagaaggttaaagatgcagtcaaaagattcaggactaattgcatcaagaacacagagaaagacatatttctcaagatcagaagtactattccagtatggacgattcaaggcttgcttcataaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcctactgaatctaaggccatgcatggagtctaagattcaaatcgaggatctaacagaactcgccgtgaagactggcgaacagttcatacagagtcttttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacactctggtctactccaaaaatgtcaaagatacagtctcagaagaccaaagggctattgagacttttcaacaaaggataatttcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaaggacagtagaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggctatcattcaagatctctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgacatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacgctcgagtataagagctctatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattacatttacaattaccatggggcgcgccccttgtcctcaaccaaatctcgccatcggtcttacgtcgcactcggattttggcggtttgacaatcctccttcaaatcaacgaagtggaaggattacagataaaaagagaggggacatggatttcagtcaaacctctacctaatgcgttcgtagtgaatgttggagatattttggagataatgactaatggaatttaccatagtgtcgatcaccgggcagtagtaaactcaacaaatgagaggctctcaatcgcaacatttcatgaccctagtctagagtcggtaataggcccaatatcaagcttgattactccagagacacctgctttgtttaaaagtggaatttaaatccccagatgaacatggcatcgtggtgattgatgaaactgctgctgtcggctttaacctctctttaggcattggtttcgaagcgggcaacaagccgaaagaactgtacagcgaagaggcagtcaacggggaaactcagcaagcgcacttacaggcgattaaagagctgatagcgcgtgacaaaaaccacccaagcgtggtgatgtggagtattgccaacgaaccggatacccgtccgcaaggtgcacgggaatatttcgcgccactggcggaagcaacgcgtaaactcgacccgacgcgtccgatcacctgcgtcaatgtaatgttctgcgacgctcacaccgataccatcagcgatctctttgatggggatcctccacttttaaacaaagcaggtgtctctggagtaatcaagcttgatattgggcctattaccgactctagactagggtcatgaaatgttgcgattgagagcctctcatttgttgagtttactactgcccggtgatcgacactatggtaaattccattagtcattatctccaaaatatctccaacattcactacgaacgcattaggtagaggtttgactgaaatccatgtcccctctctttttatctgtaatccttccacttcgttgatttgaaggaggattgtcaaaccgccaaaatccgagtgcgacgtaagaccgatggcgagatttggttgaggacaaggactagtccctagagtcctgctttaatgagatatgcgagacgcctatgatcgcatgatatttgctttcaattctgttgtgcacgttgtaaaaaacctgagcatgtgtagctcagatccttaccgccggtttcggttcattctaatgaatgaatatatcacccgttactatcgtatttttatgaataatattctccgttcaatttactgattgtaccctactacttatatgtacaatattaaaatgaaaacaatatattgtgctgaataggtttatagcgacatctatgatagagcgccacaataacaaacaattgcgttttattattacaaatccaattttaaaaaaagcggcagaaccggtcaaacctaaaagactgattacataaatcttattcaaatttcaaaagtgccccaggggctagtatctacgacacaccgagcggcgaactaataacgctcactgaagggaactccggttccccgccggcgcgcatgggtgagattccttgaagttgagtattggccgtccgctctaccgaaagttacgggcaccattcaacccggtccagcacggcggccgggtaaccgacttgctgccccgagaattatgcagcatttttttggtgtatgtgggccccaaatgaagtgcaggtcaaaccttgacagtgacgacaaatcgttgggcgggtccagggcgaattttgcgacaacatgtcgaggctcagcagggcgatcgcagacgtcgggatcttctgcaagcatctctatttcctgaaggtctaacctcgaagatttaagatttaattacgtttataattacaaaattgattctagtatctttaatttaatgcttatacattattaattaatttagtactttcaatttgttttcagaaattattttactattttttataaaataaaagggagaaaatggctatttaaatactagcctattttatttcaattttagcttaaaatcagccccaattagccccaatttcaaattcaaatggtccagcccaattcctaaataacccacccctaacccgcccggtttccccttttgatccatgcagtcaacgcccagaatttccctatataattttttaattcccaaacacccctaactctatcccatttctcaccaaccgccacatagatctatcctcttatctctcaaactctctcgaaccttcccctaaccctagcagcctctcatcatcctcacctcaaaacccaccggggccggccatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggaggccggttctttttgtcaagaccgacctgtccggtgccctgaatgaacttcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgactcatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaggcgcgccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatccctagggaagttcctattccgaagttcctattctctgaaaagtataggaacttctttgcgtattgggcgctcttggcctttttggccaccggtcgtacggttaaaaccaccccagtacattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgccaccagccagccaacagctccccgaccggcagctcggcacaaaatcaccactcgatacaggcagcccatcagtcc SEQ ID NO: 6gcgatcgctccaatcccacaaaaatctgagcttaacagcacagttgctcctctcagagcagaatcgggtattcaacaccctcatatcaactactacgttgtgtataacggtccacatgccggtatatacgatgactggggttgtacaaaggcggcaacaaacggcgttcccggagttgcacacaagaaatttgccactattacagaggcaagagcagcagctgacgcgtacacaacaagtcagcaaacagacaggttgaacttcatccccaaaggagaagctcaactcaagcccaagagctttgctaaggccctaacaagcccaccaaagcaaaaagcccactggctcacgctaggaaccaaaaggcccagcagtgatccagccccaaaagagactcctttgccccggagattacaatggacgatttcctctatctttacgatctaggaaggaagttcgaaggtgaaggtgacgacactatgttcaccactgataatgagaaggttagcctcttcaatttcagaaagaatgctgacccacagatggttagagaggcctacgcagcaggtctcatcaagacgatctacccgagtaacaatctccaggagatcaaataccttcccaagaaggttaaagatgcagtcaaaagattcaggactaattgcatcaagaacacagagaaagacatatttctcaagatcagaagtactattccagtatggacgattcaaggcttgcttcataaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcctactgaatctaaggccatgcatggagtctaagattcaaatcgaggatctaacagaactcgccgtgaagactggcgaacagttcatacagagtcttttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacactctggtctactccaaaaatgtcaaagatacagtctcagaagaccaaagggctattgagacttttcaacaaaggataatttcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaaggacagtagaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggctatcattcaagatctctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgacatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacgctcgagtataagagctctatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattacatttacaattaccatggggcgcgccccttgtcctcaaccaaatctcgccatcggtcttacgtcgcactcggattttggcggtttgacaatcctccttcaaatcaacgaagtggaaggattacagataaaaagagaggggacatggatttcagtcaaacctctacctaatgcgttcgtagtgaatgttggagatattttggagataatgactaatggaatttaccatagtgtcgatcaccgggcagtagtaaactcaacaaatgagaggctctcaatcgcaacatttcatgaccctagtctagagtcggtaataggcccaatatcaagcttgattactccagagacacctgctttgtttaaaagtggaatttaaatccccagatgaacatggcatcgtggtgattgatgaaactgctgctgtcggctttaacctctctttaggcattggtttcgaagcgggcaacaagccgaaagaactgtacagcgaagaggcagtcaacggggaaactcagcaagcgcacttacaggcgattaaagagctgatagcgcgtgacaaaaaccacccaagcgtggtgatgtggagtattgccaacgaaccggatacccgtccgcaaggtgcacgggaatatttcgcgccactggcggaagcaacgcgtaaactcgacccgacgcgtccgatcacctgcgtcaatgtaatgttctgcgacgctcacaccgataccatcagcgatctctttgatggggatcctccacttttaaacaaagcaggtgtctctggagtaatcaagcttgatattgggcctattaccgactctagactagggtcatgaaatgttgcgattgagagcctctcatttgttgagtttactactgcccggtgatcgacactatggtaaattccattagtcattatctccaaaatatctccaacattcactacgaacgcattaggtagaggtttgactgaaatccatgtcccctctctttttatctgtaatccttccacttcgttgatttgaaggaggattgtcaaaccgccaaaatccgagtgcgacgtaagaccgatggcgagatttggttgaggacaaggactagtccctagagtcctgctttaatgagatatgcgagacgcctatgatcgcatgatatttgctttcaattctgttgtgcacgttgtaaaaaacctgagcatgtgtagctcagatccttaccgccggtttcggttcattctaatgaatgaatatatcacccgttactatcgtatttttatgaataatattctccgttcaatttactgattgtaccctactacttatatgtacaatattaaaatgaaaacaatatattgtgctgaataggtttatagcgacatctatgatagagcgccacaataacaaacaattgcgttttattattacaaatccaattttaaaaaaagcggcagaaccggtcaaacctaaaagactgattacataaatcttattcaaatttcaaaagtgccccaggggctagtatctacgacacaccgagcggcgaactaataacgctcactgaagggaactccggttccccgccggcgcgcatgggtgagattccttgaagttgagtattggccgtccgctctaccgaaagttacgggcaccattcaacccggtccagcacggcggccgggtaaccgacttgctgccccgagaattatgcagcatttttttggtgtatgtgggccccaaatgaagtgcaggtcaaaccttgacagtgacgacaaatcgttgggcgggtccagggcgaattttgcgacaacatgtcgaggctcagcagggcgatcgca SEQ ID NO: 7ccccttgtcctcaaccaaatctcgccatcggtcttacgtcgcactcggattttggcggtttgacaatcctccttcaaatcaacgaagtggaaggattacagataaaaagagaggggacatggatttcagtcaaacctctacctaatgcgttcgtagtgaatgttggagatattttggagataatgactaatggaatttaccatagtgtcgatcaccgggcagtagtaaactcaacaaatgagaggctctcaatcgcaacatttcatgaccctagtctagagtcggtaataggcccaatatcaagcttgattactccagagacacctgctttgtttaaaagtggaatttaaatccccagatgaacatggcatcgtggtgattgatgaaactgctgctgtcggctttaacctctctttaggcattggtttcgaagcgggcaacaagccgaaagaactgtacagcgaagaggcagtcaacggggaaactcagcaagcgcacttacaggcgattaaagagctgatagcgcgtgacaaaaaccacccaagcgtggtgatgtggagtattgccaacgaaccggatacccgtccgcaaggtgcacgggaatatttcgcgccactggcggaagcaacgcgtaaactcgacccgacgcgtccgatcacctgcgtcaatgtaatgttctgcgacgctcacaccgataccatcagcgatctctttgatggggatcctccacttttaaacaaagcaggtgtctctggagtaatcaagcttgatattgggcctattaccgactctagactagggtcatgaaatgttgcgattgagagcctctcatttgttgagtttactactgcccggtgatcgacactatggtaaattccattagtcattatctccaaaatatctccaacattcactacgaacgcattaggtagaggtttgactgaaatccatgtcccctctctttttatctgtaatccttccacttcgttgatttgaaggaggattgtcaaaccgccaaaatccgagtgcgacgtaagaccgatggcgagatttggttgaggacaagg SEQ ID NO: 8ccttgtcctcaaccaaatctcgccatcggtcttacgtcgcactcggattttggcggtttgacaatcctccttcaaatcaacgaagtggaaggattacagataaaaagagaggggacatggatttcagtcaaacctctacctaatgcgttcgtagtgaatgttggagatattttggagataatgactaatggaatttaccatagtgtcgatcaccgggcagtagtaaactcaacaaatgagaggctctcaatcgcaacatttcatgaccctagtctagagtcggtaataggcccaatatcaagcttgattactccagagacacctgctttgtttaaaagtgga SEQ ID NO: 9 TAACCGACTTGCTGCCCCGASEQ ID NO: 10 AAATAGAGATGCTTGCAGAAGATCCCG SEQ ID NO: 11CGTTTGAATCTTGCTGGCCGTGAT SEQ ID NO: 12 TAGACGAGCTGCCTTTGGAAGTGTSEQ ID NO: 13: CGTCTTGCGCACTGATTTGAA SEQ ID NO 14:CGTTTGAATCTTGCTGGCCGTGAT SEQ ID NO: 15 (T6ODM)guucuuaauu cauuaauuaa uuuagaaaaa ucauggagaa agcaaaacuu augaagcuag   60guaaugguau ggaaauacca aguguucaag aauuggcuaa acucacgcuu gccgaaauuc  120caucucgaua cguaugcgcc aaugaaaacc uuuuguugcc uaugggugca ucugucauaa  180augaucauga aaccauuccu gucaucgaua uagaaaauuu auuaucucca gaaccaauaa  240ucggaaaguu agaauuagau aggcuucauu uugcuugcaa agaauggggu uuuuuucagg  300uagugaacca uggagucgac gcuucauugg uggauagugu aaaaucagaa auucaagguu  360ucuuuaaccu uucuauggau gagaaaacua aauaugaaca ggaagaugga gauguggaag  420gauuuggaca aggcuuuauu gaaucagagg accaaacacu ugauugggca gauauauuua  480ugauguucac ucuuccacuc cauuuaagga agccucacuu auuuucaaaa cucccagugc  540cucucaggga gacaaucgaa uccuacucau cagaaaugaa aaaguuaucc augguucucu  600uuaauaagau ggaaaaagcu cuacaaguac aagcagccga gauuaagggu augucagagg  660uguuuauaga ugggacacaa gcaaugagga ugaacuauua ucccccuugu ccucaaccaa  720aucucgccau cggucuuacg ucgcacucgg auuuuggcgg uuugacaauc cuccuucaaa  780ucaacgaagu ggaaggauua cagauaaaaa gagaggggac auggauuuca gucaaaccuc  840uaccuaaugc guucguagug aauguuggag auauuuugga gauaaugacu aauggaauuu  900accauagugu cgaucaccgg gcaguaguaa acucaacaaa ugagaggcuc ucaaucgcaa  960cauuucauga cccuagucua gagucgguaa uaggcccaau aucaagcuug auuacuccag 1020agacaccugc uuuguuuaaa aguggaucua cauaugggga ucuuguggag gaauguaaaa 1080caaggaagcu cgauggaaaa ucauuucuug acuccaugag gauuugaaaa cucaagaaaa 1140aauaauacga cgugauugca ugucagauuc aacuauccuu uugucguuuu uuggugcucg 1200aguccuuaau uguuuugauc auugcuuuug auucuaauua auaagacuuu ucucaagaac 1260cacauguaau guaccuuuac uuucagaaaa uaaaaaguau ugaggcacaa augagaaaau 1320ugagagagug cuugagaagu guaauuucuc gaaagugcgu uguguuugaa aaaaaaaaaa 1380aaaaaa                                                            1386SEQ ID NO: 16 (CODM)guaaagauug auauaugauc ugaagaucug acaagaaagu ucaucaaaua uagaguucau   60ggagacacca auacuuauca agcuaggcaa ugguuuguca auaccaagug uucaggaauu  120ggcuaaacuc acgcuugcag aaauuccauc ucgauacaca ugcaccggug aaagcccguu  180gaauaauauu ggugcgucug uaacagauga ugaaacaguu ccugucaucg auuugcaaaa  240uuuacuaucu ccagaacccg uaguuggaaa guuagaauug gauaagcuuc auucugcuug  300caaagaaugg gguuucuuuc agcugguuaa ccauggaguc gacgcuuuac ugauggacaa  360uauaaaauca gaaauuaaag guuucuuuaa ccuuccaaug aaugagaaaa cuaaauacgg  420acagcaagau ggagauuuug aaggauuugg acaacccuau auugaaucgg aggaccaaag  480acuugauugg acugaagugu uuagcauguu aagucuuccu cuccauuuaa ggaagccuca  540uuuguuucca gaacucccuc ugccuuucag ggagacacug gaauccuacc uaucaaaaau  600gaaaaaacua ucaacgguug ucuuugagau guuggaaaaa ucucuacaau uaguugagau  660uaaagguaug acagacuuau uugaagaugg guugcaaaca augaggauga acuauuaucc  720uccuuguccu cgaccagagc uuguacuugg ucuuacguca cacucggauu uuagcgguuu  780gacaauucuc cuucaacuua augaaguuga aggauuacaa auaagaaaag aagagaggug  840gauuucaauc aaaccucuac cugaugcguu cauagugaau guuggagaca uuuuggagau  900aaugacuaau gggauuuacc guagcgucga gcaccgggca guaguaaacu caacaaagga  960gaggcucuca aucgcgacau uucaugacuc uaaacuagag ucagaaauag gcccaauuuc 1020gagcuugguc acaccagaga caccugcuuu guucaaaaga gguagguaug aggauauuuu 1080gaaggaaaau cuuucaagga agcuugaugg aaaaucauuu cucgacuaca ugaggaugug 1140agaaagugug aacauauauu auacuccaca uuguguuuaa uauaugauga aauaaguugc 1200uuuugaagua ugaugaaaua aguugguuuu gaagaauuca uauugugcuu aaauuucgug 1260gaugacugag agauuuauua uguaauaaua auguauuggu uugaagauuc ucgucucacu 1320auauguaaga cucuguuugg gucaagugau guaaucacgg uugaaauaag uugcuuuuga 1380agaauucaua uggugcuuaa uauuauguaa uaaauaaugu auuggauuga aaaaaaaaaa 1440aaaaaaaaaa aa                                                     1452

1. A method of increasing accumulation of thebaine in an opium poppyplant, the method comprising genetically modifying the genome of theplant to include one or more stable genetic modifications tosimultaneously reduce the activity of thebaine 6-O-demethylase (T6ODM)and codeine 3-O-demethylase (CODM) in the poppy plant.
 2. The method ofclaim 1, wherein T6ODM has the amino acid sequence of SEQ ID NO: 1, CODMhas the amino acid sequence of SEQ ID NO: 3, or T6ODM has the amino acidsequence of SEQ ID NO: 1 and CODM has the amino acid sequence of SEQ IDNO:
 3. 3. (canceled)
 4. The method of claim 1, wherein geneticallymodifying the plant to simultaneously reduce the activity of T6ODM andCODM comprises: introducing an expression construct to reduce theaccumulation of transcripts from an endogenous gene encoding T6ODM,optionally wherein the sequence of the expression construct to reducethe accumulation of transcripts from the endogenous gene encoding T6ODMcomprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4; geneticallymodifying the plant to introduce a loss of function mutation in anendogenous gene encoding T6ODM; introducing an expression construct toreduce the accumulation of transcripts from an endogenous gene encodingCODM, optionally wherein the sequence of the expression construct toreduce the accumulation of transcripts from the endogenous gene encodingCODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4; geneticallymodifying the plant to introduce a loss of function mutation in anendogenous gene encoding CODM; introducing an expression construct toreduce the accumulation of transcripts from an endogenous gene encodingT6ODM, optionally wherein the sequence of the expression construct toreduce the accumulation of transcripts from the endogenous gene encodingT6ODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4, andintroducing an expression construct to reduce the accumulation oftranscripts from an endogenous gene encoding CODM, optionally whereinthe sequence of the expression construct to reduce the accumulation oftranscripts from the endogenous gene encoding CODM comprises a portionof SEQ ID NO: 2 or SEQ ID NO: 4; genetically modifying the plant tointroduce a loss of function mutation in an endogenous gene encodingT6ODM and introducing an expression construct to reduce the accumulationof transcripts from an endogenous gene encoding CODM, optionally whereinthe sequence of the expression construct to reduce the accumulation oftranscripts from the endogenous gene encoding CODM comprises a portionof SEQ ID NO: 2 or SEQ ID NO: 4; introducing an expression construct toreduce the accumulation of transcripts from an endogenous gene encodingT6ODM, optionally wherein the sequence of the expression construct toreduce the accumulation of transcripts from the endogenous gene encodingT6ODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4, andgenetically modifying the plant to introduce a loss of function mutationin an endogenous gene encoding CODM; or genetically modifying the plantto introduce a loss of function mutation in an endogenous gene encodingT6ODM and a loss of function mutation in an endogenous gene encodingCODM. 5.-9. (canceled)
 10. A genetically modified opium poppy planthaving reduced activity of thebaine 6-O-demethylase (T6ODM) and-codeine3-O-demethylase (CODM) relative to a wild type plant, wherein thegenetically modified opium poppy plant comprises one or more stablegenetic modifications to reduce expression of T6ODM, CODM, or both. 11.The plant of claim 10 comprising a first expression construct forreducing the expression of T6ODM, optionally wherein the firstexpression construct comprises a first nucleic acid molecule encoding afirst hairpin RNA for reducing expression of an endogenous gene encodingT6ODM; and a second expression construct for reducing expression ofCODM, optionally wherein the second expression construct comprises asecond nucleic acid molecule encoding a second hairpin RNA for reducingexpression of an endogenous gene encoding CODM.
 12. (canceled)
 13. Theplant ell of claim 11, wherein the endogenous gene encoding T6ODMencodes an mRNA comprising the sequence of SEQ ID NO: 15 and theendogenous gene encoding CODM encodes an mRNA comprising the sequence ofSEQ ID NO: 16; or the endogenous gene encoding T6ODM encodes an mRNAcomprising the sequence of SEQ ID NO: 15 and the endogenous geneencoding CODM encodes an mRNA comprising the sequence of SEQ ID NO: 16.14. The plant of claim 13, wherein the nucleic acid molecule encodingthe first hairpin RNA comprises a portion of SEQ ID NO: 2 and thenucleic acid molecule encoding the second hairpin RNA comprises aportion of SEQ ID NO: 4; or the nucleic acid molecule encoding the firsthairpin RNA comprises a portion of SEQ ID NO: 2 and the nucleic acidmolecule encoding the second hairpin RNA comprises a portion of SEQ IDNO:
 4. 15.-17. (canceled)
 18. The plant of claim 10, comprising anexpression construct comprising a nucleic acid molecule for reducing theexpression of T6ODM and CODM, optionally wherein the nucleic acidmolecule encodes: a hairpin RNA for reducing expression of an endogenousgene encoding CODM; a hairpin RNA for reducing expression of anendogenous gene encoding T6ODM; or a single hairpin RNA sufficient toreduce expression of endogenous genes encoding T6ODM and CODM. 19.-21.(canceled)
 22. The plant of claim 18, wherein the nucleic acid moleculecomprises a portion of SEQ ID NO:2, SEQ ID NO:4, or both.
 23. The plantof claim 18, wherein the expression construct comprises a first nucleicacid molecule encoding a first hairpin RNA for reducing expression of anendogenous gene encoding T6ODM and a second nucleic acid moleculeencoding a second hairpin RNA for reducing expression of an endogenousgene encoding CODM, optionally wherein each of the first nucleic acidmolecule and the second nucleic acid molecule comprise a portion of SEQID NO: 2, SEQ ID NO: 4, or both.
 24. The plant of claim 23, wherein theendogenous gene encoding T6ODM encodes an mRNA comprising the sequenceof SEQ ID NO: 15 the endogenous gene encoding T6ODM encodes apolypeptide having the sequence of SEQ ID NO 1, the endogenous geneencoding CODM encodes an mRNA comprising the sequence of SEQ ID NO: 16,the endogenous gene encoding CODM encodes a polypeptide having thesequence of SEQ ID NO 3, or any combination thereof. 25.-28. (canceled)29. The plant of claim 18, wherein the nucleic acid molecule encodingthe hairpin RNA comprises a portion of SEQ ID NO: 7 or SEQ ID NO:
 8. 30.(canceled)
 31. The plant of claim 11, wherein the first nucleic acidmolecule comprises a portion of SEQ ID NO: 7 or SEQ ID NO:
 8. 32.(canceled)
 33. The plant of claim 11, wherein the second nucleic acidmolecule comprises a portion of SEQ ID NO: 7 or SEQ ID NO:
 8. 34. Theplant of claim 31, wherein the second nucleic acid molecule comprises aportion of SEQ ID NO: 7 or SEQ ID NO:
 8. 35. The plant cell of claim 10,wherein the plant is genetically modified to have reduced activity ofT6ODM, and wherein reduced activity of CODM is conferred by a mutationin the endogenous gene encoding CODM that was not introduced by geneticmodification of the plant, optionally wherein the mutation in theendogenous gene encoding CODM that was not introduced by geneticmodification of the plant is the mutation present in seeds of the plantdeposited under Patent Deposit Designation PTA-9109, or the plant isgenetically modified to have reduced activity of CODM, and whereinreduced activity of T6ODM is conferred by a mutation in the endogenousgene encoding T6ODM that was not introduced by genetic modification ofthe plant, or the mutation in the endogenous gene encoding T6ODM thatwas not introduced by genetic modification of the plant is the mutationpresent in seeds of the plant deposited under Patent Deposit DesignationPTA-9110. 36.-95. (canceled)
 96. An isolated nucleic acid molecule,wherein the sequence of the nucleic acid molecule comprises SEQ ID NO:7.97. An expression vector for simultaneously reducing the expression ofendogenous genes encoding thebaine 6-O-demethylase (T6ODM) and codeine3-O-demethylase (CODM) in an opium poppy plant, the expression vectorcomprising an isolated nucleic acid as defined in claim
 96. 98.(canceled)
 99. (canceled)
 100. Poppy straw from a plant as defined inclaim
 10. 101. Latex from a plant as defined in claim 10.